Antibody-drug conjugates comprising substituted benzo[e]pyrrolo[1,2-α][1,4]diazepines

ABSTRACT

A novel antibody-pyrrolodiazepine derivative and a novel antibody-pyrrolodiazepine derivative conjugate using the same, comprising substituted benzo[e]pyrrolo[1,2-a][1,4]diazepines represented by the [Formula 24], [Formula 25], [Formula 26], and/or [Formula 27].

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 37 U.S.C. § 371 toInternational Patent Application No. PCT/JP2018/036252, filed Sep. 28,2018, which claims priority to and the benefit of Japanese PatentApplication No. 2017-190713, filed on Sep. 29, 2017. The contents ofthese applications are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, is named 122763-0102_SL.txtand is 113 kb in size.

TECHNICAL FIELD

The present invention relates to an antibody-drug conjugate useful as anantitumor drug, the antibody-drug conjugate having an antibody capableof targeting tumor cells and a pyrrolobenzodiazepine derivative that areconjugated to each other via a linker structure moiety.

BACKGROUND ART

Antibody-drug conjugates (ADCs) have a drug with cytotoxic activityconjugated to an antibody that binds to an antigen expressed on thesurface of cancer cells and is capable of cellular internalization ofthe antigen through the binding. ADCs can effectively deliver the drugto cancer cells, and are thus expected to cause accumulation of the drugwithin the cancer cells and to kill the cancer cells.

For example, the ADC Adcetris (TM) (brentuximab vedotin), which hasmonomethyl auristatin E conjugated to an anti-CD30 monoclonal antibody,has been approved as a therapeutic drug for Hodgkin's lymphoma andanaplastic large-cell lymphoma. Kadcyla (TM) (trastuzumab emtansine),which has emtansine conjugated to an anti-HER2 monoclonal antibody, isused for treatment of HER2-positive advanced and recurrent breastcancers.

A useful example of drugs to be conjugated for ADCs ispyrrolobenzodiazepine (PBD). PBD exhibits cytotoxicity, for example, bybinding to the PuGPu sequence in the DNA minor groove. Anthramycin, anaturally-occurring PBD, was first discovered in 1965, and since thisdiscovery various naturally-occurring PBDs and analog PBDs thereof havebeen discovered (Non Patent Literatures 1 to 4).

The general structural formula of PBDs is represented by the followingformula:

Known are PBDs different in the number of, types of, and sites ofsubstituents in the A and C ring parts, and those different in degree ofunsaturation in the B and C ring parts.

PBDs are known to come to have dramatically enhanced cytotoxicitythrough formation of a dimer structure (Non Patent Literatures 5, 6),and various ADCs with a dimer PBD have been reported (Patent Literatures1 to 13). However, a PBD having a spiro ring at its C2-position and anADC form thereof have not known.

Human CLDN6 (claudin-6, hereinafter expressed as hCLDN6), a member ofclaudin (CLDN) family proteins, is a four-transmembrane proteinconsisting of 220 amino acid residues. Previous studies have suggestedthat hCLDN6 is overexpressed in some cancers, and is an attractivecancer therapeutic target (Non Patent Literatures 7 to 9). CLDN familyproteins are incorporated into cells by endocytosis, and some of thefamily proteins have been reported to have short turnover time (NonPatent Literature 10), and hence CLDN family proteins are considered tobe suitable as the target of antibody-drug conjugates (ADCs).

From such information suggesting the relation to cancer, monoclonalantibodies capable of specifically recognizing hCLDN6 have beendiscovered (Patent Literatures 14, 15), and ADCs having monomethylauristatin E (MMAE) or maytansinoid (DM1), which are tubulinpolymerization inhibitors, conjugated to a CLDN6-specific monoclonalantibody have been reported (Non Patent Literature 11).

On the other hand, antibodies capable of recognizing multiple members ofthe CLDN family are considered to allow a wider range of application oftreatment, and in view of this an ADC having a pyrrolobenzodiazepine(PBD) with potent cytocidal effect conjugated to an antibody capable ofrecognizing CLDN6 and CLDN9 (Patent Literature 16) has been disclosed(Patent Literature 17).

However, the intensities of activity of the ADCs are still insufficient,and there exist unmet medical needs for use of hCLDN6 as a therapeutictarget.

CITATION LIST Patent Literature

-   Patent Literature 1: WO 2013/173496-   Patent Literature 2: WO 2014/130879-   Patent Literature 3: WO 2017/004330-   Patent Literature 4: WO 2017/004025-   Patent Literature 5: WO 2017/020972-   Patent Literature 6: WO 2016/036804-   Patent Literature 7: WO 2015/095124-   Patent Literature 8: WO 2015/052322-   Patent Literature 9: WO 2015/052534-   Patent Literature 10: WO 2016/115191-   Patent Literature 11: WO 2015/052321-   Patent Literature 12: WO 2015/031693-   Patent Literature 13: WO 2011/130613-   Patent Literature 14: WO 2009/087978-   Patent Literature 15: WO 2011/057788-   Patent Literature 16: WO 2015/069794-   Patent Literature 17: WO 2017/096163

Non Patent Literature

-   Non Patent Literature 1: Julia Mantaj, et al., Angewandte Chemie    International Edition 2016, 55, 2-29-   Non Patent Literature 2: Dyeison Antonow. et al., Chemical Reviews    2010, 111, 2815-2864-   Non Patent Literature 3: In Antibiotics III. Springer Verlag, New    York, pp. 3-11-   Non Patent Literature 4: Accounts of Chemical Research 1986, 19, 230-   Non Patent Literature 5: Journal of the American Chemical Society    1992, 114, 4939-   Non Patent Literature 6: Journal of Organic Chemistry 1996, 61, 8141-   Non Patent Literature 7: BMC Cancer, 2006, 6, 186.-   Non Patent Literature 8: Histopatholody, 2012, 61, 1043-1056.-   Non Patent Literature 9: Int J Cancer, 2014, 135, 2206-2214.-   Non Patent Literature 10: J Membrane Biol, 2004, 199, 29-38.-   Non Patent Literature 11: 14th Annu Meet Cancer Immunother (CIMT)    (May 10-12, Mainz) 2016, Abst 185

SUMMARY OF INVENTION Problems to be Resolved by the Invention

The present invention provides a novel antibody-pyrrolobenzodiazepine(PBD) derivative conjugate and a novel pyrrolobenzodiazepine (PBD)derivative.

The present invention provides a novel anti-CLDN6 antibody.

In addition, the present invention provides a pharmaceutical compositioncontaining the antibody-PBD derivative conjugate, PBD derivative, oranti-CLDN6 antibody with antitumor activity.

Further, the present invention provides a method for treating cancer byusing the antibody-PBD derivative conjugate, PBD derivative, oranti-CLDN6 antibody.

Means of Solving the Problems

The present inventors diligently examined to find that a novelantibody-pyrrolobenzodiazepine (PBD) derivative conjugate has strongantitumor activity, thereby completing the present invention.

Specifically, the present invention relates to the following.

[1] An antibody-drug conjugate represented by the following formula:

wherein

m¹ represents an integer of 1 to 10, preferably an integer of 2 to 8;

Ab represents an antibody or a functional fragment of the antibody,where the antibody optionally has a remodeled glycan;

L represents a linker linking Ab and D;

Ab may bond directly via its amino acid residue to L, or may bond via aglycan or a remodeled glycan of Ab to L; and

D represents a drug represented by the following formula:

wherein

the asterisk represents bonding to L;

n¹ represents an integer of 2 to 8;

A represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring or three- to five-membered saturated heterocycleoptionally substituted with one to four halogen atoms;

R¹ and R² each independently represent a C1 to C6 alkoxy group, a C1 toC6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1 toC6 alkylthio group, a halogen atom, or —NR′R″, wherein R′ and R″ eachindependently represent a hydrogen atom or a C1 to C6 alkyl group;

R³, R⁴, and R⁵ are selected from (i) to (iii):

(i) R³ and R⁴ are combined, together with the carbon atoms to which R³and R⁴ are bound, to form a double bond, and R⁵ represents an aryl groupor heteroaryl group optionally having one or more substituents selectedfrom group 1 or a C1 to C6 alkyl group optionally having one or moresubstituents selected from group 2,

(ii) R³ represents a hydrogen atom, and R⁴ and R⁵ are combined, togetherwith the carbon atom to which R⁴ and R⁵ are bound, to form a three- tofive-membered saturated hydrocarbon ring or a three- to five-memberedsaturated heterocycle, or CH₂═, and

(iii) R³, R⁴, and R⁵ are combined, together with the carbon atom towhich R³ is bound and the carbon atom to which R⁴ and R⁵ are bound, toform a benzene ring or six-membered heterocycle optionally having one ormore substituents selected from group 3;

R⁶ and R⁷ each represent a hydrogen atom, or R⁶ and R⁷ are combined torepresent an imine bond (C═N);

R⁸ represents a hydroxy group or a C1 to C3 alkoxy group;

X and Y each independently represent an oxygen atom, a nitrogen atom, ora sulfur atom;

group 1 represents:

a) a C1 to C6 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C6 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR′, —NR′R″,—C(═NR′)—NR″R′″, and —NHC(═NR′)—NR″R′″,

c) a halogen atom,

d) a C3 to C5 cycloalkoxy group,

e) a C1 to C6 alkylthio group,

f) —NR′R″,

g) —C(═NR′)—NR″R′″,

h) —NHC(═NR′)—NR″R′″,

i) —NHCOR′, or

j) a hydroxy group,

wherein R′ and R″ are as defined above, and R′″ each independentlyrepresents a hydrogen atom or a C1 to C6 alkyl group; group 2 representsa halogen atom, a hydroxy group, or a C1 to C6 alkoxy group; and

group 3 represents a halogen atom, or a C1 to C6 alkyl group or C1 to C6alkoxy group optionally substituted with one to three halogen atoms.

[2] The antibody-drug conjugate according to [1], wherein

A represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R¹ and R² each independently represent a C1 to C3 alkoxy group;

R³ and R⁴ are combined together with the carbon atoms to which R³ and R⁴are bound to form a double bond;

R⁵ represents an aryl group or heteroaryl group optionally having one ormore substituents selected from group 4, or a C1 to C3 alkyl groupoptionally having one or more substituents selected from group 5;

X and Y are each an oxygen atom;

group 4 represents:

a) a C1 to C3 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C3 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR″,—C(═NR′)—NR″R′″, and —NHC(═NR′)—NR″R′″,

c) a C3 to C5 cycloalkoxy group,

d) —C(═NR′)—NR″R′″,

e) —NHC(═NR′)—NR″R′″, or

f) a hydroxy group,

wherein R′, R″, and R′″ each independently represent a hydrogen atom ora C1 to C3 alkyl group; and

group 5 represents a halogen atom, a hydroxy group, or a C1 to C3 alkoxygroup.

[3] The antibody-drug conjugate according to [1], wherein

A represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R¹ and R² each independently represent a C1 to C3 alkoxy group;

R³ represents a hydrogen atom;

R⁴ and R⁵ are combined, together with the carbon atom to which

R⁴ and R⁵ are bound, to form a three- to five-membered saturatedhydrocarbon ring, or ═CH₂; and

X and Y are each an oxygen atom.

[4] The antibody-drug conjugate according to [1], wherein

A represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R¹ and R² each independently represent a C1 to C3 alkoxy group;

R³, R⁴, and R⁵ are combined, together with the carbon atom to which R³is bound and the carbon atom to which R⁴ and R⁵ are bound, to form abenzene ring optionally having one or more substituents selected fromgroup 6;

X and Y are each an oxygen atom; and

group 6 represents a halogen atom, or a C1 to C3 alkyl group or C1 to C3alkoxy group optionally substituted with one to three halogen atoms.

[5] The antibody-drug conjugate according to [1] or [2], wherein D isrepresented by any one of the following two formulas:

wherein each asterisk represents bonding to L.

[6] The antibody-drug conjugate according to [1] or [3], wherein D isrepresented by any one of the following two formulas:

wherein each asterisk represents bonding to L.

[7] The antibody-drug conjugate according to any one of [1] to [6],wherein

L is represented by -Lb-La-Lp-NH—B—CH₂—O(C═O)—*, the asteriskrepresenting bonding to D;

B represents a phenyl group or a heteroaryl group;

Lp represents a linker consisting of an amino acid sequence cleavable ina target cell;

La represents any one selected from the group:

—C(═O)—(CH₂CH₂)n²-C(═O)—, —C(═O)—(CH₂CH₂)n²-C(═O)—NH—(CH₂CH₂)n³-C(═O)—,

—C(═O)—(CH₂CH₂)n²-C(═O)—NH—(CH₂CH₂O)n³-CH₂—C(═O)—,—C(═O)—(CH₂CH₂)n²-NH—C(═O)—(CH₂CH₂O)n³-CH₂CH₂—C(═O)—, and—(CH₂)n⁴-O—C(═O)—;

n² represents an integer of 1 to 3, n³ represents an integer of 1 to 5,and n⁴ represents an integer of 0 to 2; and

Lb represents a spacer bonding La and a glycan or remodeled glycan ofAb.

[8] The antibody-drug conjugate according to [7], wherein B is any oneselected from a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridylgroup, a 2,5-pyrimidyl group, and a 2,5-thienyl group.

[9] The antibody-drug conjugate according to [8], wherein B is a1,4-phenyl group.

[10] The antibody-drug conjugate according to any one of [7] to [9],wherein Lp is amino acid residues composed of two to seven amino acids.

[11] The antibody-drug conjugate according to any one of [7] to [10],wherein Lp is amino acid residues consisting of amino acids selectedfrom glycine, valine, alanine, phenylalanine, glutamic acid, isoleucine,proline, citrulline, leucine, serine, lysine, and aspartic acid.

[12] The antibody-drug conjugate according to any one of [7] to [11],wherein Lp is selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,and -GGPL-.

[13] The antibody-drug conjugate according to any one of [7] to [12],wherein La is selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, and—OC(═O)—.

[14] The antibody-drug conjugate according to any one of [7] to [13],wherein Lb is represented by the following formula:

wherein, in each structural formula for Lb shown above,

each asterisk represents bonding to La, and each wavy line representsbonding to a glycan or remodeled glycan of Ab.

[15] The antibody-drug conjugate according to any one of [7] to [14],wherein

L is represented by -Lb-La-Lp-NH—B—CH₂—O(C═O)—*, wherein

B is a 1,4-phenyl group;

Lp represents any one selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-;

La represents any one selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, and—OC(═O)—; and

Lb is represented by the following formula:

wherein, in each structural formula for Lb shown above,

each asterisk represents bonding to La, and each wavy line representsbonding to a glycan or remodeled glycan of Ab.

[16] The antibody-drug conjugate according to any one of [7] to [15],wherein

L is selected from the following group:

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GG-(D-)VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGPI-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVK-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGPL-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)-VA-NH—B—CH₂—OC(═O)—,

—Z²—OC(═O)-GGVA-NH—B—CH₂—OC(═O)—, and—Z³—CH₂—OC(═O)-GGVA-NH—B—CH₂—OC(═O)—, wherein

Z¹ represents the following structural formula:

Z² represents the following structural formula:

and

Z³ represents the following structural formula:

wherein, in each structural formula for Z¹, Z², and Z³,

each asterisk represents bonding to La, each wavy line representsbonding to a glycan or remodeled glycan of Ab; and

B represents a 1,4-phenyl group.

[17] The antibody-drug conjugate according to [16], wherein

L is selected from the following group:

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—, and

—Z¹—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)-VA-NH—B—CH₂—OC(═O)—,wherein

B is a 1,4-phenyl group, and Z¹ represents the following structuralformula:

wherein, in the structural formula for Z¹,

each asterisk represents bonding to C(═O) neighboring to Z¹, and eachwavy line represents bonding to a glycan or remodeled glycan of Ab.

[18] The antibody-drug conjugate according to any one of [1] to [6],wherein

L is represented by -Lb-La-Lp-NH—B—CH₂—O(C═O)—*, wherein

the asterisk represents bonding to D;

B represents a 1,4-phenyl group;

Lp represents -GGVA- or -VA;

La represents —(CH₂)n⁹-C(═O)— or—(CH₂CH₂)n¹⁰-C(═O)—NH—(CH₂CH₂O)n¹¹-CH₂CH₂—C(═O)—, wherein n⁹ representsan integer of 2 to 7, n¹⁰ represents an integer of 1 to 3, and n¹represents an integer of 6 to 10; and

Lb is -(succinimid-3-yl-N)—.

[19] The antibody-drug conjugate according to [18], wherein

L represents any one selected from the following group:

-(succinimid-3-yl-N)—(CH₂)₅—C(═O)—VA-NH—B—CH₂—OC(═O)—,

-(succinimid-3-yl-N)—(CH₂)₅—C(═O)-GGVA-NH—B—CH₂—OC(═O)—, and

-(succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₉—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,wherein B is a 1,4-phenyl group.

[20] The antibody-drug conjugate according to any one of [1] to [19],wherein the antibody is IgG.

[21] The antibody-drug conjugate according to [20], wherein the antibodyis IgG1, IgG2, or IgG4.

[22] The antibody-drug conjugate according to any one of [1] to [21],wherein the antibody binds to a tumor cell, and is incorporated andinternalizes in the tumor cell.

[23] The antibody-drug conjugate according to [22], wherein the antibodyfurther has antitumor effect.

[24] The antibody-drug conjugate according to any one of [1] to [17] and[20] to [23], wherein the antibody bonds via a glycan bonding to Asn297of the antibody (N297 glycan) to L.

[25] The antibody-drug conjugate according to [24], wherein the N297glycan is a remodeled glycan.

[26] The antibody-drug conjugate according to [24] or [25], wherein theN297 glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, orN297-(Fuc)SG, with N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SGhaving structures represented by the following formulas:

wherein

the wavy line represents bonding to Asn297 of the antibody;

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end is bound via an amide bond to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in the 1-3branched chain of β-Man in the N297 glycan;

the asterisk represents bonding to linker L; and

n⁵ represents an integer of 2 to 10,

wherein

the wavy line represents bonding to Asn297 of the antibody;

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end is bound via an amide bond to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in the 1-6branched chain of β-Man in the N297 glycan;

the asterisk represents bonding to linker L; and

n⁵ represents an integer of 2 to 10, and

wherein

the wavy line represents bonding to Asn297 of the antibody;

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end is bound via an amide bond to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in each of the1-3 and 1-6 branched chains of β-Man in the N297 glycan;

the asterisk represents bonding to linker L; and

n⁵ represents an integer of 2 to 10.

[27] The antibody-drug conjugate according to [26], wherein n⁵ is aninteger of 2 to 5.

[28] The antibody-drug conjugate according to any one of [24] to [27],represented by the following formula:

wherein

m² represents an integer of 1 or 2;

L is a linker linking the N297 glycan of Ab and D, and being any oneselected from the following group:

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—, and

—Z¹—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)-VA-NH—B—CH₂—OC(═O)—,wherein

B is a 1,4-phenyl group, and Z¹ represents the following structuralformula:

wherein, in the structural formulas for Z¹,

each asterisk represents bonding to C(═O) neighboring to Z¹, and eachwavy line represents bonding to the N297 glycan of Ab;

Ab represents an IgG antibody or a functional fragment of the antibody;

the N297 glycan of Ab represents any one of N297-(Fuc)MSG1,N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, withN297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG having structuresrepresented by the following formulas:

wherein

each wavy line represents bonding to Asn297 of the antibody,

L(PEG) in the N297 glycan represents —NH—CH₂CH₂—(O—CH₂CH₂)n⁵-*, wherein

n⁵ represents an integer of 2 to 5, the amino group at the left end isbound via an amide bond to carboxylic acid at the 2-position of a sialicacid at the non-reducing terminal in each or either one of the 1-3 and1-6 branched chains of β-Man in the N297 glycan, and each asteriskrepresents bonding to a nitrogen atom at the 1- or 3-position of thetriazole ring of Z¹ in linker L; and

D is any one selected from the following group:

wherein

each asterisk represents bonding to L.

[29] An antibody-drug conjugate selected from the following group:

wherein, in each structural formula shown above,

m² represents an integer of 1 or 2;

Ab represents an IgG antibody or a functional fragment of the antibody;

the N297 glycan of Ab represents any one of N297-(Fu)MSG1,N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, withN297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG having structuresrepresented by the following formulas:

wherein

each wavy line represents bonding to Asn297 of the antibody,

L(PEG) in the N297 glycan represents —NH—CH₂CH₂—(O—CH₂CH₂)₃—*, wherein

the amino group at the left end is bound via an amide bond to carboxylicacid at the 2-position of a sialic acid at the non-reducing terminal ineach or either one of the 1-3 and 1-6 branched chains of β-Man in theN297 glycan, and each asterisk represents bonding to a nitrogen atom atthe 1- or 3-position of the triazole ring in the correspondingstructural formula.

[30] An antibody which binds to CLDN6 and/or CLDN9, or a functionalfragment of the antibody.

[31] The antibody according to [30] or a functional fragment of theantibody, wherein CLDN6 is a molecule consisting of an amino acidsequence represented by SEQ ID NO: 1, and CLDN9 is a molecule consistingof an amino acid sequence represented by SEQ ID NO: 3.

[32] The antibody according to [30] or [31] or a functional fragment ofthe antibody, the antibody comprising a heavy chain comprising CDRH1,CDRH2, and CDRH3 and a light chain comprising CDRL1, CDRL2, and CDRL3 asdescribed in any one of the following (a) and (b):

(a) CDRH1 consisting of an amino acid sequence represented by SEQ ID NO:9, CDRH2 consisting of an amino acid sequence represented by SEQ ID NO:10, and CDRH3 consisting of an amino acid sequence represented by SEQ IDNO: 11, and CDRL1 consisting of an amino acid sequence represented bySEQ ID NO: 5, CDRL2 consisting of an amino acid sequence represented bySEQ ID NO: 6, and CDRL3 consisting of an amino acid sequence representedby SEQ ID NO: 7 or an amino acid sequence having one or two amino acidsubstitutions in the amino acid sequence represented by SEQ ID NO: 7;and

(b) CDRH1 consisting of an amino acid sequence represented by SEQ ID NO:15, CDRH2 consisting of an amino acid sequence represented by SEQ ID NO:16, and CDRH3 consisting of an amino acid sequence represented by SEQ IDNO: 17, and CDRL1 consisting of an amino acid sequence represented bySEQ ID NO: 12, CDRL2 consisting of an amino acid sequence represented bySEQ ID NO: 13, and CDRL3 consisting of an amino acid sequencerepresented by SEQ ID NO: 14.

[33] The antibody according to [32] or a functional fragment of theantibody, the antibody comprising a heavy chain comprising CDRH1, CDRH2;and CDRH3 and a light chain comprising CDRL1, CDRL2, and CDRL3 asdescribed in any one of the following (a) and (b):

(a) CDRH1 consisting of an amino acid sequence represented by SEQ ID NO:9, CDRH2 consisting of an amino acid sequence represented by SEQ ID NO:10, and CDRH3 consisting of an amino acid sequence represented by SEQ IDNO: 11, and CDRL1 consisting of an amino acid sequence represented bySEQ ID NO: 5, CDRL2 consisting of an amino acid sequence represented bySEQ ID NO: 6, and CDRL3 consisting of an amino acid sequence representedby SEQ ID NO: 7 or an amino acid sequence represented by SEQ ID NO: 8;and

(b) CDRH1 consisting of an amino acid sequence represented by SEQ ID NO:15, CDRH2 consisting of an amino acid sequence represented by SEQ ID NO:16, and CDRH3 consisting of an amino acid sequence represented by SEQ IDNO: 17, and CDRL1 consisting of an amino acid sequence represented bySEQ ID NO: 12, CDRL2 consisting of an amino acid sequence represented bySEQ ID NO: 13, and CDRL3 consisting of an amino acid sequencerepresented by SEQ ID NO: 14.

[34] The antibody according to any one of [30] to [33] or a functionalfragment of the antibody, the antibody comprising a heavy chain variableregion and a light chain variable region as described in any one of thefollowing (a) and (b):

(a) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 21 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 19; and

(b) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 25 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 23.

[35] The antibody according to any one of [30] to [34] or a functionalfragment of the antibody, the antibody comprising a heavy chain variableregion consisting of an amino acid sequence selected from the groupconsisting of the following (a) to (e) and a light chain variable regionconsisting of an amino acid sequence selected from the group consistingof the following (f) to (k):

(a) an amino acid sequence represented by SEQ ID NO: 54;

(b) an amino acid sequence represented by SEQ ID NO: 58;

(c) an amino acid sequence represented by SEQ ID NO: 62;

(d) an amino acid sequence with a homology of at least 95% or higher toa sequence of a framework region excluding CDR sequences in any of thesequences (a) to (c);

(e) an amino acid sequence having one to several amino acid deletions,substitutions, or additions in a sequence of a framework regionexcluding CDR sequences in any of the sequences (a) to (c);

(f) an amino acid sequence represented by SEQ ID NO: 38;

(g) an amino acid sequence represented by SEQ ID NO: 42;

(h) an amino acid sequence represented by SEQ ID NO: 46;

(i) an amino acid sequence represented by SEQ ID NO: 50;

(j) an amino acid sequence with a homology of at least 95% or higher toa sequence of a framework region excluding CDR sequences in any of thesequences (f) to (i); and

(k) an amino acid sequence having one to several amino acid deletions,substitutions, or additions in a sequence of a framework regionexcluding CDR sequences in any of the sequences (f) to (i).

[36] The antibody according to [35] or a functional fragment of theantibody, the antibody comprising a heavy chain variable region and alight chain variable region selected from the group consisting of thefollowing (a) to (e):

(a) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 54 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 38;

(b) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 58 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 42;

(c) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 54 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 46;

(d) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 58 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 50; and

(e) a heavy chain variable region consisting of an amino acid sequencerepresented by SEQ ID NO: 62 and a light chain variable regionconsisting of an amino acid sequence represented by SEQ ID NO: 46.

[37] The antibody according to any one of [30] to [36] or a functionalfragment of the antibody, wherein the antibody is a chimeric antibody.

[38] The antibody according to any one of [30] to [36] or a functionalfragment of the antibody, wherein the antibody is a humanized antibody.

[39] The antibody according to any one of [30] to [38] or a functionalfragment of the antibody, the antibody comprising a heavy chain constantregion of human IgG1, human IgG2, or human IgG4.

[40] The antibody according to [38] or [39] or a functional fragment ofthe antibody, the antibody comprising a heavy chain and a light chainselected from the group consisting of the following (a) to (e):

(a) a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 52 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 234 of SEQ ID NO: 36 (H1L1);

(b) a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 56 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 234 of SEQ ID NO: 40 (H2L2);

(c) a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 52 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 234 of SEQ ID NO: 44 (H1L3);

(d) a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 56 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 234 of SEQ ID NO: 48 (H2L4); and

(e) a heavy chain consisting of an amino acid sequence consisting ofamino acid residues 20 to 471 of SEQ ID NO: 60 and a light chainconsisting of an amino acid sequence consisting of amino acid residues21 to 234 of SEQ ID NO: 44 (H3L3).

[41] The antibody according to [30] or [31] or a functional fragment ofthe antibody, wherein the antibody binds to a site of an antigenrecognizable to the antibody according to any one of [32] to [36] and[40].

[42] The antibody according to [30] or [31] or a functional fragment ofthe antibody, wherein the antibody competes with the antibody accordingto any one of [32] to [36] and [40] for binding to CLDN6 and/or CLDN9.

[43] A polynucleotide encoding the antibody according to any one of [30]to [42].

[44] The polynucleotide according to [43], comprising a polynucleotideselected from the group consisting of the following (a) to (j):

(a) a polynucleotide encoding a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 54 and apolynucleotide encoding a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 38;

(b) a polynucleotide encoding a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 58 and apolynucleotide encoding a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 42;

(c) a polynucleotide encoding a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 54 and apolynucleotide encoding a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 46;

(d) a polynucleotide encoding a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 58 and apolynucleotide encoding a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 50;

(e) a polynucleotide encoding a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 62 and apolynucleotide encoding a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 46;

(f) a polynucleotide encoding a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 52and a polynucleotide encoding a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 36;

(g) a polynucleotide encoding a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 56and a polynucleotide encoding a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 40;

(h) a polynucleotide encoding a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 52and a polynucleotide encoding a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 44;

(i) a polynucleotide encoding a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 56and a polynucleotide encoding a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 48;and

(j) a polynucleotide encoding a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 60and a polynucleotide encoding a light chain consisting of an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 44.

[45] An expression vector comprising the polynucleotide according to[43] or [44].

[46] A host cell transformed with the expression vector according to[45].

[47] The host cell according to [46], wherein the host cell is aeukaryotic cell.

[48] The host cell according to [47], wherein the host cell is an animalcell.

[49] A method for producing the antibody according to any one of [30] to[42] or a functional fragment of the antibody, the method comprising thesteps of: culturing the host cell according to any one of [46] to [48];and collecting a targeted antibody from the culture obtained in the stepof culturing.

[50] An antibody obtained by using the method according to [49], or afunctional fragment of the antibody.

[51] The antibody according to any one of [30] to [42] and [50] or afunctional fragment of the antibody, the antibody comprising one or twoor more modifications selected from the group consisting of N-linkedglycosylation, O-linked glycosylation, N-terminal processing, C-terminalprocessing, deamidation, isomerization of aspartic acid, oxidation ofmethionine, addition of a methionine residue at an N terminus, amidationof a proline residue, and deletion of one or two amino acid residues atthe carboxyl terminus of a heavy chain.

[52] The antibody according to [51] or a functional fragment of theantibody, wherein one or several amino acid residues are deleted at thecarboxyl terminus of a heavy chain.

[53] The antibody according to [52] or a functional fragment of theantibody, wherein one amino acid residue is deleted at the carboxylterminus of each of the two heavy chains.

[54] The antibody according to any one of [50] to [53] or a functionalfragment of the antibody, wherein a proline residue at the carboxylterminus of a heavy chain is further amidated.

[55] A method for producing a glycan-remodeled antibody, the methodcomprising the steps of:

i) culturing the host cell according to any one of [46] to [48] andcollecting a targeted antibody from the culture obtained;

ii) treating the antibody obtained in step i) with hydrolase to producea (Fucα1,6)GlcNAc-antibody; and

iii)-1 reacting the (Fucα1,6)GlcNAc-antibody and a glycan donnermolecule in the presence of transglycosidase, the glycan donner moleculeobtained by introducing a PEG linker having an azide group to thecarbonyl group of carboxylic acid at the 2-position of a sialic acid inMSG (9) or SG (10) and oxazolinating the reducing terminal, or

iii)-2 reacting the (Fucα1,6)GlcNAc-antibody and a glycan donnermolecule in the presence of transglycosidase, the glycan donner moleculeobtained by introducing a PEG linker having an azide group to thecarbonyl group of carboxylic acid at the 2-position of a sialic acid in(MSG-)Asn or (SG-)Asn with an α-amino group optionally protected and tothe carbonyl group of carboxylic acid in the Asn, causing action ofhydrolase, and then oxazolinating the reducing terminal.

[56] The method according to [55], further comprising the step ofpurifying the (Fucα1,6)GlcNAc-antibody through purification of areaction solution in step ii) with a hydroxyapatite column.

[57] A method for producing the antibody-drug conjugate according to anyone of [1] to [29], the method comprising the steps of:

i) producing a glycan-remodeled antibody by using the method accordingto [55] or [56]; and

ii) reacting a drug-linker having DBCO (a production intermediate) andan azide group in a glycan of the glycan-remodeled antibody made in stepi).

[58] A glycan-remodeled antibody obtained by using the method accordingto [55] or [56].

[59] An antibody-drug conjugate obtained by using the method accordingto [57].

[60] An antibody-drug conjugate selected from the following group:

wherein, in each structural formula shown above,

m² represents an integer of 1 or 2;

Ab represents the antibody according to any one of [30] to [42], [50] to[54], and [58] or a functional fragment of the antibody, or an anti-HER2antibody; and

the N297 glycan of Ab represents any one of N297-(Fuc)MSG1,N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, withN297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG having structuresrepresented by the following formulas:

wherein

each wavy line represents bonding to Asn297 of the antibody,

L(PEG) represents —NH—CH₂CH₂—(O—CH₂CH₂)₃—*, wherein the amino group atthe left end is bound via an amide bond to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in each oreither one of the 1-3 and 1-6 branched chains of β-Man in the N297glycan, and each asterisk represents bonding to a nitrogen atom at the1- or 3-position of the triazole ring in the corresponding structuralformula.

[61] The antibody-drug conjugate according to any one of [1] to [29],[59], and [60], wherein the average number of conjugated drug moleculesper antibody molecule in the antibody-drug conjugate is 1 to 3 or 3 to5.

[62] A compound, a salt of the compound, or a hydrate of the compound orthe salt, the compound represented by the following formula:

wherein

l represents an integer of 2 to 8;

E represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring or three- to five-membered saturated heterocycleoptionally substituted with one to four halogen atoms;

R⁹ and R¹⁰ each independently represent a C1 to C6 alkoxy group, a C1 toC6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1 toC6 alkylthio group, a halogen atom, or —NR′R″, wherein

R′ and R″ each independently represent a hydrogen atom or a C1 to C6alkyl group;

R¹¹, R¹² and R¹³ are selected from the following (i) to (iii):

(i) R¹¹ and R¹² are combined, together with the carbon atoms to whichR¹¹ and R¹² are bound, to form a double bond, and R¹³ represents an arylgroup or heteroaryl group optionally having one or more substituentsselected from group 7 or a C1 to C6 alkyl group optionally having one ormore substituents selected from group 8,

(ii) R¹¹ represents a hydrogen atom, and R¹² and R¹³ are combinedtogether to form a three- to five-membered saturated hydrocarbon ring ora three- to five-membered saturated heterocycle, or CH₂═, and

(iii) R¹¹ and R¹² are combined together to form a benzene ring orsix-membered heterocycle optionally having one or more substituentsselected from group 9, and R¹³ represents a single bond;

R¹⁴ and R¹⁵ each represent a hydrogen atom, or R¹⁴ and R¹⁵ are combinedto represent an imine bond (C═N);

R¹⁶ and R¹⁷ represent any one of the following (a) and (b):

(a) R¹⁶ and R¹⁷ are combined to form an imine bond (N═C), and

(b) R¹⁶ represents J-La′-Lp′-NH—B′—CH₂—O (C═O)—*,

wherein

the asterisk represents bonding to the nitrogen atom neighboring to R¹⁶,

B′ represents a phenyl group or a heteroaryl group,

Lp′ represents a linker consisting of an amino acid sequence cleavablein a target cell,

La′ represents any one of the following group:

—C(═O)—(CH₂CH₂)n⁶-C(═O)—, —C(═O)—(CH₂CH₂)n⁶-C(═O)—NH—(CH₂CH₂)n⁷-C(═O)—,

—C(═O)—(CH₂CH₂)n⁶-C(═O)—NH—(CH₂CH₂O)n⁷-CH₂—C(═O)—,

—C(═O)—(CH₂CH₂)n⁶-NH—C(═O)—(CH₂CH₂O)n⁷-CH₂CH₂—C(═O)—, —(CH₂)n⁸-O—C(═O)—,

—(CH₂)n¹²-C(═O)—, and —(CH₂CH₂)n¹³-C(═O)—NH—(CH₂CH₂O)n¹⁴-CH₂CH₂—C(═O)—,wherein n⁶ represents an integer of 1 to 3, n⁷ represents an integer of1 to 5, n⁸ represents an integer of 0 to 2, n¹² represents an integer of2 to 7, n¹³ represents an integer of 1 to 3, and n¹⁴ represents aninteger of 6 to 10,

J represents any one of the following:

wherein, in the structural formulas for J shown above,

each asterisk represents bonding to La′;

R¹⁷ represents a hydroxy group or a C1 to C3 alkoxy group;

V and W are each independently an oxygen atom, a nitrogen atom, or asulfur atom;

group 7 represents:

a) a C1 to C6 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C6 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR′, —NR′R″,—C(═NR′)—NR″R′″, and —NHC(═NR′)—NR″R′″,

c) a halogen atom,

d) a C3 to C5 cycloalkoxy group,

e) a C1 to C6 alkylthio group,

f) —NR′R″,

g) —C(═NR′)—NR″R′″,

h) —NHC(═NR′)—NR″R′″,

i) —NHCOR′, or

j) a hydroxy group,

wherein

R′ and R″ are as defined above, and R′″ each independently represents ahydrogen atom or a C1 to C6 alkyl group;

group 8 represents a halogen atom, a hydroxy group, or a C1 to C6 alkoxygroup; and

group 9 represents a halogen atom or a C1 to C6 alkyl group or a C1 toC6 alkoxy group optionally substituted with one to three halogen atoms.

[63] The compound according to [62], a salt of the compound, or ahydrate of the compound or the salt, wherein

E represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R⁹ and R¹⁰ each independently represent a C1 to C3 alkoxy group;

R¹¹ and R¹² are combined together with the carbon atoms to which R¹¹ andR¹² are bound to form a double bond;

R¹³ represents an aryl group or heteroaryl group optionally having oneor more substituents selected from group 10, or a C1 to C3 alkyl groupoptionally having one or more substituents selected from group 11;

V and W are each an oxygen atom;

group 10 represents:

a) a C1 to C3 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C3 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR″,—C(═NR′)—NR″R′″, and —NHC(═NR′)—NR″R′″,

c) a C3 to C5 cycloalkoxy group,

d) —C(═NR′)—NR″R′″,

e) —NHC(═NR′)—NR″R′″, or

f) a hydroxy group,

wherein

R′, R″, and R′″ each independently represent a hydrogen atom or a C1 toC3 alkyl group; and

group 11 represents a halogen atom, a hydroxy group, or a C1 to C3alkoxy group.

[64] The compound according to [62], a salt of the compound, or ahydrate of the compound or the salt, wherein

E represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R⁹ and R¹⁰ each independently represent a C1 to C3 alkoxy group;

R¹¹ represents a hydrogen atom;

R¹² and R¹³ are combined, together with the carbon atom to which R¹² andR¹³ are bound, to form a three- to five-membered saturated hydrocarbonring, or ═CH₂; and

V and W are each an oxygen atom.

[65] The compound according to [62], a salt of the compound, or ahydrate of the compound or the salt, wherein

E represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring optionally substituted with one or two halogen atoms;

R⁹ and R¹⁰ each independently represent a C1 to C3 alkoxy group;

R¹¹, R¹², and R¹³ are combined, together with the carbon atom to whichR¹¹ is bound and the carbon atom to which R¹² and R¹³ are bound, to forma benzene ring optionally having one or more substituents selected fromgroup 12;

V and W are each an oxygen atom; and

group 12 represents a halogen atom or a C1 to C3 alkyl group or a C1 toC3 alkoxy group optionally substituted with one to three halogen atoms.

[66] The compound according to any one of [62] to [65], a salt of thecompound, or a hydrate of the compound or the salt, wherein

B′ is any one selected from a 1,4-phenyl group, a 2,5-pyridyl group, a3,6-pyridyl group, a 2,5-pyrimidyl group, and a 2,5-thienyl group.

[67] The compound according to [66], a salt of the compound, or ahydrate of the compound or the salt, wherein B′ is a 1,4-phenyl group.

[68] The compound according to any one of [62] to [67], a salt of thecompound, or a hydrate of the compound or the salt, wherein Lp′ is aminoacid residues selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-,-GG(D-)P-I-, and -GGPL-.

[69] The compound according to any one of [62] to [68], a salt of thecompound, or a hydrate of the compound or the salt, wherein La′ isselected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, —OC(═O)—,

—(CH₂)₅—C(═O)—, and —CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—.

[70] The compound according to any one of [62] to [69], a salt of thecompound, or a hydrate of the compound or the salt, wherein

R¹⁶ is represented by J-La′-Lp′-NH—B′—CH₂—O (C═O)—*, wherein

B′ is a 1,4-phenyl group;

Lp′ represents any one selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, and -GGPL-;

La′ represents any one selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, —OC(═O)—,

—(CH₂)₅—C(═O)—, and —CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—; and

J represents any one of the following:

wherein, in the structural formulas for J,

each asterisk represents bonding to La′.

[71] The compound according to any one of [62] to [70], a salt of thecompound, or a hydrate of the compound or the salt, wherein

R¹⁶ is selected from the following group:

J¹-C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GG-(D-)VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGPI-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGFG-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVK-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGPL-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)-VA-NH—B′—CH₂—OC(═O)—,

J²-OC(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J³-CH₂—OC(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)—VA-NH—B′—CH₂—OC(═O)—, andJ⁴-CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—, wherein

J¹, J², J³, and J⁴ represent the following structural formulas:

wherein, in the structural formulas for J¹, J², J³, and J⁴, eachasterisk represents bonding to a neighboring group, and

B′ is a 1,4-phenyl group.

[72] The compound according to any one of [62] to [71], a salt of thecompound, or a hydrate of the compound or the salt, wherein

R¹⁶ is selected from the following group:

J¹-C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)-VA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—, and

J⁴-(CH₂)₅—C(═O)—VA-NH—B′—CH₂—OC(═O)—, wherein

B′ is a 1,4-phenyl group, and

J¹ and J⁴ represent the following structural formulas:

wherein, in the structural formulas for J¹ and J⁴,

each asterisk represents bonding to a neighboring group.

[73] A compound, a salt of the compound, or a hydrate of the compound orthe salt, wherein the compound is any one compound selected from thefollowing formulas:

[74] A compound, a salt of the compound, or a hydrate of the compound orthe salt, wherein the compound is any one compound selected from thefollowing formulas:

[75] The antibody-drug conjugate according to any one of [1] to [29] and[59] to [61], wherein D is represented by the following formula:

[76] The compound according to any one of [62] to [72] and [74], a saltof the compound, or a hydrate of the compound or the salt, the compoundrepresented by the following formula:

[77] The antibody-drug conjugate according to any one of [1] to [29] and[59] to [61], wherein D is represented by the following formula:

[78] The compound according to any one of [62] to [72] and [74], a saltof the compound, or a hydrate of the compound or the salt, the compoundrepresented by the following formula:

[79] A pharmaceutical composition comprising any of the antibody-drugconjugate according to any one of [1] to [29] and [59] to [61], [75] and[77], a salt of the antibody-drug conjugate, or a hydrate of theantibody-drug conjugate or the salt; the antibody according to any oneof [30] to [42], [50] to [54], and [58] or a functional fragment of theantibody; and the compound according to any one of [62] to [74], [76]and [78], a salt of the compound, or a hydrate of the compound or thesalt.

[80] The pharmaceutical composition according to [79], being anantitumor drug.

[81] The pharmaceutical composition according to [80], wherein the tumoris a tumor expressing CLDN6 and/or CLDN9.

[82] The pharmaceutical composition according to [80] or [81], whereinthe tumor is ovarian cancer (surface epithelial tumor, stromal tumor, orgerm cell tumor), lung cancer (non-small cell lung cancer or small celllung cancer), gastric cancer, endometrial cancer, testicular cancer(seminoma, or non-seminoma), uterine cervix cancer, placentalchoriocarcinoma, kidney cancer, urothelial cancer, colorectal cancer,prostate cancer, glioblastoma multiforme, brain tumor, pancreaticcancer, breast cancer, melanoma, liver cancer, bladder cancer, oresophageal cancer.

[83] A method for treating a tumor, wherein any of the antibody-drugconjugate according to any one of [1] to [29] and [59] to [61], [75] and[77], a salt of the antibody-drug conjugate, or a hydrate of theantibody-drug conjugate or the salt; the antibody according to any oneof [30] to [42], [50] to [54], and [58] or a functional fragment of theantibody; and the compound according to any one of [62] to [74], [76]and [78], a salt of the compound, or a hydrate of the compound or thesalt is administered to an individual.

[84] The method according to [83], wherein the tumor is a tumorexpressing CLDN6 and/or CLDN9.

[85] The method according to [83] or [84], wherein the tumor is ovariancancer (surface epithelial tumor, stromal tumor, or germ cell tumor),lung cancer (non-small cell lung cancer or small cell lung cancer),gastric cancer, endometrial cancer, testicular cancer (seminoma ornon-seminoma), uterine cervix cancer, placental choriocarcinoma, kidneycancer, urothelial cancer, colorectal cancer, prostate cancer,glioblastoma multiforme, brain tumor, pancreatic cancer, breast cancer,melanoma, liver cancer, bladder cancer, or esophageal cancer.

[86] A method for treating a tumor, wherein a pharmaceutical compositioncomprising at least one selected from the antibody-drug conjugateaccording to any one of [1] to [29] and [59] to [61], [75] and [77], asalt of the antibody-drug conjugate, or a hydrate of the antibody-drugconjugate or the salt; the antibody according to any one of [30] to[42], [50] to [54], and [58] or a functional fragment of the antibody;and the compound according to any one of [62] to [74], [76] and [78], asalt of the compound, or a hydrate of the compound or the salt, and atleast one antitumor drug are administered to an individualsimultaneously, separately, or consecutively.

[87] A compound exhibiting proton NMR having peak positionssubstantially similar to peak positions listed in Table 1 or Table 2.

Advantageous Effects of Invention

The novel antibody-pyrrolobenzodiazepine (PBD) derivative conjugateprovided by the present invention is superior in antitumor activity andsafety, and hence useful as an antitumor agent. The PBD derivative ofthe present invention has antitumor activity, and thus is useful as adrug for the conjugate. In addition, the antibody of the presentinvention recognizes tumor cells or binds to tumor cells, and hence isuseful as an antibody for the conjugate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of the drug-conjugate of the presentinvention (the molecule of (I)). (a) indicates drug D, (b) indicateslinker L, (c) indicates N₃-L(PEG)-, and (d) indicates N297 glycan (openellipse: NeuAc(Sia), open hexagon: Man, filled hexagon: GlcNAc, opendiamond: Gal, open inverted triangle: Fuc). (b) and (c) are combinedtogether to form a triazole ring by reaction between the azide group(filled teardrop shape) of (c) and the spacer (open semicircle) of (b).The Y-shaped diagram represents antibody Ab. For convenience, in thisschematic diagram, N297 glycan is indicated as N297-(Fuc)MSG and thediagram shows an embodiment wherein any one of two branches in each ofN297 glycans has a sialic acid to which a PEG linker having an azidegroup (N₃-L(PEG)-) bonds while other branch has no sialic acid at thenon-reducing terminal (i.e. N297-(Fuc)MSG); however, another embodimentwherein each of two branches of N297 glycan has a sialic acid to which aPEG linker having an azide group bonds at the non-reducing terminal(i.e. N297-(Fuc)SG) is also acceptable. Unless otherwise stated, such amanner of illustration is applied throughout the present specification.

FIGS. 2A and 2B are schematic diagrams illustrating the structures of a(Fucα1,6)GlcNAc-antibody (the molecule of FIG. 2A in (II) of FIGS. 2Aand 2B), which is a production intermediate of the drug-conjugate of thepresent invention, and an MSG-type glycan-remodeled antibody (themolecule of (III) in FIG. 2B of FIGS. 2A and 2B). In each of thediagrams, the Y-shaped diagram represents antibody Ab as in FIG. 1 . InFIG. 2A, (e) indicates N297 glycan consisting only of GlcNAc at the6-position connected to 1-positions of Fuc via an a glycosidic bond. InFIG. 2B, (d) indicates the same N297 glycan as in FIG. 1 , and (f)indicates a structure of a PEG linker portion having an azide group,specifically, an azide group to be bonded to liker L at the end. Thebonding mode of the PEG linker having an azide group is as described forFIG. 1 .

FIGS. 3A and 3B are schematic diagrams for the step of producing anMSG-type glycan-remodeled antibody from an antibody produced in ananimal cell. As in FIGS. 2A and 2B, molecules (II) and (III) in thisFigure represent an (Fucα1,6)GlcNAc-antibody and an MSG-typeglycan-remodeled antibody, respectively. Molecule (IV) is an antibodyproduced in an animal cell, and is a mixture of molecules withheterogeneous N297 glycan moieties. FIG. 3A illustrates the step ofproducing homogeneous (Fucα1,6)GlcNAc-antibody (II) by treatingheterogeneous N297 glycan moieties of (IV) with hydrolase such as EndoS.FIG. 3B illustrates the step of producing the MSG-type glycan-remodeledantibody of (III) by subjecting GlcNAc of N297 glycan in antibody (II)to transglycosidase such as an EndoS D233Q/Q303L variant totransglycosylate the glycan of an MSG-type glycan donor molecule. TheMSG-type glycan donor molecule used here has a sialic acid at thenon-reducing terminal of MSG modified with a PEG linker having an azidegroup. Thus, resulting MSG-type N297 glycan-remodeled antibody also hasa sialic acid at the non-reducing terminal modified in the same manneras described for FIG. 2B. For convenience, FIG. 3B shows MSG as a donormolecule. However, a glycan-remodeled antibody in which a linkermolecule having an azide group bonds to each non-reducing terminal ofN297 glycan also can be synthesized as the remodeled antibody of (III)by using SG (10) as a glycan donor.

FIG. 4 shows the effects of the anti-HER2 antibody-drug conjugatesADC26, ADC19, and ADC54 on subcutaneously transplanted NCI-N87 cells, ahuman gastric cancer cell line.

FIG. 5 shows the effects of the anti-HER2 antibody-drug conjugate ADC49,trastuzumab, and the anti-LPS antibody-drug conjugate ADC53 onsubcutaneously transplanted NCI-N87 cells, a human gastric cancer cellline.

FIG. 6 shows the effects of the anti-HER2 antibody-drug conjugate ADC49,the anti-LPS antibody-drug conjugate ADC53, and trastuzumab-tesirine(Reference Example 1) on subcutaneously transplanted KPL-4 cells, ahuman breast cancer cell line.

FIG. 7 shows the effect of the anti-HER2 antibody-drug conjugate ADC49and trastuzumab-tesirine (Reference Example 1) on subcutaneouslytransplanted JIMT-1 cells, a human breast cancer cell line.

FIG. 8 shows the effects of the anti-CLDN6 antibody-drug conjugate ADC40and an anti-CLDN6 antibody (H1L1)-tesirine (Reference Example 1) onsubcutaneously transplanted OV-90 cells, a human ovarian cancer cellline.

FIG. 9 shows the effects of the anti-CLDN6 antibody-drug conjugate ADC40and an anti-CLDN6 antibody (H1L1)-tesirine (Reference Example 1) onsubcutaneously transplanted NIH:OVCAR-3 cells, a human ovarian cancercell line.

FIG. 10 shows the effects of the anti-TROP2 antibody-drug conjugateADC50 and the anti-LPS antibody-drug conjugate ADC53 on subcutaneouslytransplanted FaDu cells, a human head-and-neck cancer cell line.

FIG. 11 shows the full-length amino acid sequence of human CLDN6 (SEQ IDNO: 1) and the nucleotide sequence of full-length cDNA for human CLDN6(SEQ ID NO: 2).

FIG. 12 shows the full-length amino acid sequence of human CLDN9 (SEQ IDNO: 3) and the nucleotide sequence of full-length cDNA for human CLDN9(SEQ ID NO: 4).

FIG. 13 shows the amino acid sequences of CDRL1 to 3 of a B1 antibodylight chain (SEQ ID NOs: 5 to 7).

FIG. 14 shows the amino acid sequence of CDRL3 of the humanized B1antibody light chain L4 (SEQ ID NO: 8).

FIG. 15 shows the amino acid sequences of CDRH1 to 3 of a B1 antibodyheavy chain (SEQ ID NOs: 9 to 11).

FIG. 16 shows the amino acid sequences of CDRL1 to 3 of a C7 antibodylight chain (SEQ ID NOs: 12 to 14).

FIG. 17 shows the amino acid sequences of CDRH1 to 3 of a C7 antibodyheavy chain (SEQ ID NOs: 15 to 17).

FIG. 18 shows the nucleotide sequence of cDNA encoding the variableregion of a B1 antibody light chain (SEQ ID NO: 18) and the amino acidsequence of the variable region of a B1 antibody light chain (SEQ ID NO:19). Each underline in the amino acid sequence indicates a CDR sequence.

FIG. 19 shows the nucleotide sequence of cDNA encoding the variableregion of a B1 antibody heavy chain (SEQ ID NO: 20) and the amino acidsequence of the variable region of a B1 antibody heavy chain (SEQ ID NO:21). Each underline in the amino acid sequence indicates a CDR sequence.

FIG. 20 shows the nucleotide sequence of cDNA encoding the variableregion of a C7 antibody light chain (SEQ ID NO: 22) and the amino acidsequence of the variable region of a C7 antibody light chain (SEQ ID NO:23). Each underline in the amino acid sequence indicates a CDR sequence.

FIG. 21 shows the nucleotide sequence of cDNA encoding the variableregion of a C7 antibody heavy chain (SEQ ID NO: 24) and the amino acidsequence of the variable region of a C7 antibody heavy chain (SEQ ID NO:25). Each underline in the amino acid sequence indicates a CDR sequence.

FIG. 22 shows the amino acid sequence of a chB1 light chain (SEQ ID NO:28) and a DNA fragment including a DNA sequence encoding the amino acidsequence of a chB1 light chain (SEQ ID NO: 29). Each underline in theamino acid sequence indicates a CDR sequence.

FIG. 23 shows the amino acid sequence of the variable region of a chB1light chain (SEQ ID NO: 30) and the nucleotide sequence encoding a chB1light chain variable region (SEQ ID NO: 31). Each underline in the aminoacid sequence indicates a CDR sequence.

FIG. 24 shows the amino acid sequence of a chB1 heavy chain (SEQ ID NO:32) and the nucleotide sequence encoding a chB1 heavy chain (SEQ ID NO:33). Each underline in the amino acid sequence indicates a CDR sequence.

FIG. 25 shows the amino acid sequence of the variable region of a chB1heavy chain (SEQ ID NO: 34) and the nucleotide sequence encoding avariable region of a chB1 heavy chain (SEQ ID NO: 35). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 26 shows the amino acid sequence of the humanized antibody lightchain hL1 (SEQ ID NO: 36) and the nucleotide sequence encoding thehumanized antibody light chain hL1 (SEQ ID NO: 37). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 27 shows the amino acid sequence of the variable region of thehumanized antibody light chain hL1 (SEQ ID NO: 38) and the nucleotidesequence encoding the variable region of the humanized antibody lightchain hL1 (SEQ ID NO: 39). Each underline in the amino acid sequenceindicates a CDR sequence.

FIG. 28 shows the amino acid sequence of the humanized antibody lightchain hL2 (SEQ ID NO: 40) and the nucleotide sequence encoding thehumanized antibody light chain hL2 (SEQ ID NO: 41). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 29 shows the amino acid sequence of the variable region of thehumanized antibody light chain hL2 (SEQ ID NO: 42) and the nucleotidesequence encoding the variable region of the humanized antibody lightchain hL2 (SEQ ID NO: 43).

FIG. 30 shows the amino acid sequence of the humanized antibody lightchain hL3 (SEQ ID NO: 44) and the nucleotide sequence encoding thehumanized antibody light chain hL3 (SEQ ID NO: 45). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 31 shows the amino acid sequence of the variable region of thehumanized antibody light chain hL3 (SEQ ID NO: 46) and the nucleotidesequence encoding the variable region of the humanized antibody lightchain hL3 (SEQ ID NO: 47). Each underline in the amino acid sequenceindicates a CDR sequence.

FIG. 32 shows the amino acid sequence of the humanized antibody lightchain hL4 (SEQ ID NO: 48) and the nucleotide sequence encoding thehumanized antibody light chain hL4 (SEQ ID NO: 49). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 33 shows the amino acid sequence of the variable region of thehumanized antibody light chain hL4 (SEQ ID NO: 50) and the nucleotidesequence encoding the variable region of the humanized antibody lightchain hL4 (SEQ ID NO: 51). Each underline in the amino acid sequenceindicates a CDR sequence.

FIG. 34 shows the amino acid sequence of the humanized antibody heavychain hH1 (SEQ ID NO: 52) and the nucleotide sequence encoding thehumanized antibody heavy chain hH1 (SEQ ID NO: 53). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 35 shows the amino acid sequence of the variable region of thehumanized antibody heavy chain hH1 (SEQ ID NO: 54) and the nucleotidesequence encoding the variable region of the humanized antibody heavychain hH1 (SEQ ID NO: 55). Each underline in the amino acid sequenceindicates a CDR sequence.

FIG. 36 shows the amino acid sequence of the humanized antibody heavychain hH2 (SEQ ID NO: 56) and the nucleotide sequence encoding thehumanized antibody heavy chain hH2 (SEQ ID NO: 57). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 37 shows the amino acid sequence of the variable region of thehumanized antibody heavy chain hH2 (SEQ ID NO: 58) and the nucleotidesequence encoding the variable region of the humanized antibody heavychain hH2 (SEQ ID NO: 59).

FIG. 38 shows the amino acid sequence of the humanized antibody heavychain hH3 (SEQ ID NO: 60) and the nucleotide sequence encoding thehumanized antibody heavy chain hH3 (SEQ ID NO: 61). Each underline inthe amino acid sequence indicates a CDR sequence.

FIG. 39 shows the amino acid sequence of the variable region of thehumanized antibody heavy chain hH3 (SEQ ID NO: 62) and the nucleotidesequence encoding the variable region of the humanized antibody heavychain hH3 (SEQ ID NO: 63). Each underline in the amino acid sequenceindicates a CDR sequence.

FIG. 40 shows the binding abilities of a B1 antibody and a C7 antibodyto human CLDN6 and the family molecules CLDN3, CLDN4, and CLDN9 measuredby flow cytometry.

FIG. 41 shows the antibody internalization activities of a B1 antibodyand C7 antibody measured by Mab-ZAP.

FIG. 42 shows the binding abilities of the humanized anti-CLDN6antibodies H1L1, H2L2, H1L3, H2L4, and H3L3 to CLDN6 and the familymolecules measured by flow cytometry.

FIG. 43 shows the amino acid sequence of the trastuzumab light chain(SEQ ID NO: 64) and the amino acid sequence of the trastuzumab heavychain (SEQ ID NO: 65).

FIG. 44 shows the amino acid sequence of a light chain of a trastuzumabvariant (SEQ ID NO: 73) and the amino acid sequence of a heavy chain ofa trastuzumab variant (SEQ ID NO: 75).

FIG. 45 shows comparison of the amino acid sequences of chB1_H, which isa heavy chain of the chimerized human anti-CLDN6 antibody chB1, and thehumanized antibody heavy chains hH1, hH2, and hH3. The symbol “.”indicates an amino acid residue identical to the corresponding aminoacid residue of chB1_H, and each position with a symbol of an amino acidresidue indicates a substituted amino acid residue.

FIG. 46 shows comparison of the amino acid sequences of chB1_L, which isa light chain of the chimerized human anti-CLDN6 antibody chB1, and thehumanized antibody light chains hL1, hL2, hL3, and hL4. The symbol “.”indicates an amino acid residue identical to the corresponding aminoacid residue of chB1_L, and each position with symbol of an amino acidresidue indicates a substituted amino acid residue.

FIG. 47 shows the effects of the anti-HER2 antibody-drug conjugatesADC49 and ADC55 on subcutaneously transplanted KPL-4 cells, a humanbreast cancer cell line.

FIG. 48 shows the effect of the anti-HER2 antibody-drug conjugate ADC55on subcutaneously transplanted JIMT-1 cells, a human breast cancer cellline.

FIG. 49 shows the effects of the anti-HER2 antibody-drug conjugatesADC49 and ADC55, and the anti-LPS antibody-drug conjugate ADC53 onsubcutaneously transplanted CFPAC-1 cells, a human pancreatic cancercell line.

FIG. 50 shows Formula 122, which is a glycan-remodeled antibody whichmay be produced by using a method as illustrated in FIGS. 3A and 3B, forexample, according to a method described in WO 2013/120066.

FIG. 51 shows Formula 179, wherein the schematic diagram in the right ofthe structural formula represents the corresponding structure in theschematic diagram of an intermediate having a linker structure to whichan azide group has been introduced as represented by the reactionformula of Example 58.

FIG. 52 shows Formula 180, wherein the schematic diagram in the right ofthe structural formula represents the corresponding structure in theschematic diagram of an intermediate having a linker structure to whichan azide group has been introduced as represented by the reactionformula of each of Examples 60, 61, 62, 63, 64, 65, and 66.

FIG. 53 shows Formula 181, wherein the schematic diagram in the right ofthe structural formula represents the corresponding structure in theschematic diagram of an intermediate having a linker structure to whichan azide group has been introduced as represented by the reactionformula of Example 59.

FIG. 54 shows Formula 182 which represents a linker structure in whichan azide group has been introduced to a sialic acid at the non-reducingterminal of an SG-type N297 glycan. In Example 58, linker structures ofintermediates formed by introducing an azide group to an N297 glycan areall the same as the structure represented by the formula.

FIG. 55 shows Formula 183, which represents a linker structure in whichan azide group has been introduced to a sialic acid at the non-reducingterminal of an MSG-type N297 glycan. In Example 59, linker structures ofintermediates formed by introducing an azide group to an N297 glycan areall the same as the structure represented by the formula.

FIG. 56 shows Formula 184, which represents a linker structure in whichan azide group has been introduced to a sialic acid at the non-reducingterminal of an MSG1-type N297 glycan. In Example 60, linker structuresof intermediates formed by introducing an azide group to an N297 glycanare all the same as the structure represented by the formula. The sameholds true for Examples 61 to 66.

FIG. 57 shows Formula 185, Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody(H1L1). The operations same as in step 1 of Example 58 were performedusing a ca. 37.7 mg/mL anti-CLDN6 antibody solution (25 mM histidinesolution (pH 6.0), 5% sorbitol solution) prepared in Example 136 (2.5mL) to afford a 19.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1)solution (50 mM phosphate buffer (pH 6.0)) (4.8 mL).

FIG. 58 shows Formula 186, Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody(H2L2). The operations same as in step 1 of Example 58 were performedusing a ca. 20 mg/mL anti-CLDN6 antibody solution (25 mM histidinesolution (pH 6.0), 5% sorbitol solution) prepared in Example 136 (6 mL)to afford a 21.84 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2)solution (50 mM phosphate buffer (pH 6.0)) (5.7 mL).

FIG. 59 shows Formula 187, Step 1: (Fucα1,6)GlcNAc-anti-CLDN6 antibody(H1L3). The operations same as in step 1 of Example 58 were performedusing a ca. 39.4 mg/mL anti-CLDN6 antibody solution (25 mM histidinesolution (pH 6.0), 5% sorbitol solution) prepared in Example 136 (3 mL)to afford a 39.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L3)solution (50 mM phosphate buffer (pH 6.0)) (4.5 mL).

FIG. 60 shows Formula 188, Step 1: (Fucα1,6)GlcNAc-anti-CD98 antibody.The operations same as in step 1 of Example 58 were performed using aca. 20 mg/mL anti-CD98 antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) prepared in Reference Example 6 (6 mL) toafford a 21.7 mg/mL (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mMphosphate buffer (pH 6.0)) (4.7 mL).

FIG. 61 shows Formula 189, Step 1: (Fucα1,6)GlcNAc-anti-Trop2 antibody.The operations same as in step 1 of Example 58 were performed using aca. 20 mg/mL anti-Trop2 antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) obtained in Reference Example 5 (6 mL) toafford a 21.69 mg/mL (Fucα1,6)GlcNAc-anti-Trop2 antibody solution (50 mMphosphate buffer (pH 6.0)) (3.3 mL).

FIG. 62 shows Formula 190, Step 1: (Fucα1,6)GlcNAc-anti-LPS antibody.The operations same as in step 1 of Example 58 were performed using aca. 17 mg/mL anti-LPS antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) prepared in Reference Example 4 (6.6 mL) toafford a 21.03 mg/mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mMphosphate buffer (pH 6.0)) (5.4 mL).

FIG. 63 shows Formula 191, wherein the ADCs described in Examples 67 to71, 77 to 80, 82 to 88, 92 to 95, 109 to 114, and 120 were synthesized,as illustrated in the following reaction formula, by conjugating theantibody obtained in step 1 of Example 59 with a drug-linker. In theformula, R differs among drug-linkers used in those Examples.

FIG. 64 shows Formula 192, wherein the ADCs described in Examples 72,73, 75, and 91 were synthesized, as illustrated in the followingreaction formula, by conjugating the antibody obtained in step 2 ofExample 58 with a drug-linker. In the formula, R differs amongdrug-linkers used in those Examples.

FIG. 65 shows Formula 193, wherein the ADCs described in Examples 74,81, 89, 90, 96 to 105, 115, and 118 were synthesized, as illustrated inthe following reaction formula, by conjugating the antibody obtained instep 1 of Example 60 with a drug-linker. In the formula, R group differsamong drug-linkers used in those Examples.

FIG. 66 shows Formula 194, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 67 has a linker as a mixture of the two structures shown as R.

FIG. 67 shows Formula 233, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 106 has a linker as a mixture of the two structures shown as R.

FIG. 68 shows Formula 234, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 107 has a linker as a mixture of the two structures shown as R.

FIG. 69 shows Formula 235, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 108 has a linker as a mixture of the two structures shown as R.

FIG. 70 shows Formula 243, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 116 has a linker as a mixture of the two structures shown as R.

FIG. 71 shows Formula 244, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 117 has a linker as a mixture of the two structures shown as R.

FIG. 72 shows Formula 246, wherein the triazole ring to be formed instep 1 has geometric isomers, and the compound obtained in step 1 ofExample 119 has a linker as a mixture of the two structures shown as R.

DESCRIPTION OF EMBODIMENTS

The antibody-drug conjugate of the present invention is an antitumordrug having an antitumor compound conjugated via a linker structuremoiety to an antibody capable of recognizing or binding to tumor cells.

In the present invention, examples of “halogen atom” may include, butare not limited to, a fluorine atom, a chlorine atom, a bromine atom,and an iodine atom.

In the present invention, “C1 to C6 alkyl group” refers to a linear orbranched alkyl group having one to six carbon atoms. Examples of “C1 toC6 alkyl group” may include, but are not limited to, a methyl group, anethyl group, a n-propyl group, an i-propyl group, a n-butyl group, ani-butyl group, a s-butyl group, a t-butyl, a n-pentyl group, and an-hexyl.

In the present invention, “C1 to C6 alkoxy group” refers to an alkoxygroup having a linear or branched alkyl group having one to six carbonatoms. Examples of “C1 to C6 alkoxy group” may include, but are notlimited to, a methoxy group, an ethoxy group, a n-propoxy group, ani-propoxy group, a n-butoxy group, an i-butoxy, a s-butoxy group, an-pentyloxy group, and a n-hexyloxy.

In the present invention, “C1 to C6 alkylthio group” refers to analkylthio group having a linear or branched alkyl group having one tosix carbon atoms. Examples of “C1 to C6 alkylthio group” may include,but are not limited to, a methylthio group, an ethylthio group, an-propylthio group, an i-propylthio group, a n-butylthio group, ani-butylthio group, a s-butylthio group, a t-butylthio group, an-pentylthio group, and a n-hexylthio group.

In the present invention, “three- to five-membered saturated hydrocarbonring” refers to a saturated cyclic hydrocarbon group having three tofive carbon atoms. Examples of “three- to five-membered saturatedhydrocarbon ring” may include, but are not limited to, a cyclopropylgroup, a cyclobutyl group, and a cyclopentyl group.

In the present invention, “C3 to C5 cycloalkoxy group” refers to acycloalkoxy group having a saturated cyclic hydrocarbon group havingthree to five carbon atoms. Examples of “C3 to C5 cycloalkoxy group” mayinclude, but are not limited to, a cyclopropoxy group, a cyclobutoxygroup, and a cyclopentyloxy group.

In the present invention, examples of “three- to five-membered saturatedheterocycle” may include, but are not limited to, 1,3-propylene oxide,azacyclobutane, trimethylene sulfide, tetrahydrofuran, and pyrrolidine.

In the present invention, examples of “aryl group” may include, but arenot limited to, a phenyl group, a benzyl group, an indenyl group, anaphthyl group, a fluorenyl group, an anthranyl group, and aphenanthrenyl group.

In the present invention, examples of “heteroaryl group” may include,but are not limited to, a thienyl group, a pyrrolyl group, a pyrazolylgroup, a triazolyl group, an oxazolyl group, an oxadiazolyl group, athiazolyl group, a pyridyl group, a pyrimidyl group, a pyridazyl group,a pyrazinyl group, a quinolyl group, a quinoxalyl group, abenzothiophenyl group, a benzimidazolyl group, a benzotriazolyl group,and a benzofuranyl group.

In the present invention, examples of “six-membered heterocycle” mayinclude, but are not limited to, a pyridine ring, a pyrimidine ring, anda pyridazine ring.

In the present invention, “spiro-bonded” refers to the situation inwhich, as exemplified in Examples, A and a pyrrolidine ring to which Abonds, or E and a pyrrolidine ring to which E bonds form a spiro ring.

[Antibody-Drug Conjugate]

The antibody-drug conjugate of the present invention is represented bythe following formula:

m₁ represents the number of conjugated drug molecules per antibodymolecule in the antibody-drug conjugate, Ab represents an antibody or afunctional fragment of the antibody, L represents a linker linking Aband D, and D represents a drug.

<Drug>

Drug D conjugated in the antibody-drug conjugate of the presentinvention will be described. Drug D of the present invention ispreferably an antitumor compound. The antitumor compound developsantitumor effect, when a part or the entire of the linker is cleaved ina tumor cell and the antitumor compound moiety is released. When thelinker and the drug are cleaved apart at the bonding part, the antitumorcompound in the original structure is released and the originalantitumor effect is exerted.

The antitumor compound in the antibody-drug conjugate of the presentinvention is a pyrrolobenzodiazepine derivative (PBD derivative)represented by general formula (V):

Now, this will be described.

The asterisk represents bonding to linker L.

n¹ represents an integer of 2 to 8, and is preferably an integer of 2 to6, and more preferably an integer of 3 to 5.

The alkyl chain with the subscript n¹ being an integer of 2 to 8,preferably an integer of 2 to 6, and more preferably an integer of 3 to5, may include a double bond.

A represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring or a three- to five-membered saturated heterocycle, andis preferably a three- to five-membered saturated hydrocarbon ring(cyclopropane, cyclobutane, or cyclopentane), more preferablycyclopropane or cyclobutane, and most preferably cyclopropane.

The spiro-bonded three- to five-membered saturated hydrocarbon ring maybe substituted with one to four halogen atoms, and may be preferablysubstituted with one or two fluorine atoms (e.g.,2,2-difluorocyclopropane).

R¹ and R² each independently represent a C1 to C6 alkoxy group, a C1 toC6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1 toC6 alkylthio group, a halogen atom, or —NR′R″, and are each preferably aC1 to C6 alkoxy group, a C1 to C6 alkyl group, or a hydroxy group, morepreferably a C1 to C3 alkoxy group, and most preferably a methoxy group.

R³, R⁴, and R⁵ are as described in any of the following (i) to (iii).

(i) If R³ and R⁴ are combined together with the carbon atoms to which R³and R⁴ are bound to form a double bond as shown in the following:

R⁵ represents an aryl group or heteroaryl group optionally having one ormore substituents selected from group 1 or a C1 to C6 alkyl groupoptionally having one or more substituents selected from group 2, and ispreferably an aryl group optionally having one or more substituentsselected from group 1.

“Aryl group” in “aryl group or heteroaryl group optionally having one ormore substituents selected from group 1” for R⁵ is preferably a phenylgroup or a naphthyl group, and more preferably a phenyl group.

“Heteroaryl group” in “aryl group or heteroaryl group optionally havingone or more substituents selected from group 1” for R⁵ is preferably athienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, aquinoxalyl group, or a benzothiophenyl group, more preferably a2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridylgroup, or a 4-pyridyl group, and even more preferably a 3-pyridyl groupor a 3-thienyl group.

Examples of substituents of the aryl group or heteroaryl group for R⁵may include, but are not limited to, the following a) to j):

a) a C1 to C6 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C6 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR′, —NR′R″,—C(═NR′)—NR″R′, and —NHC(═NR′)—NR″R′″,

c) a halogen atom,

d) a C3 to C5 cycloalkoxy group,

e) a C1 to C6 alkylthio group,

f) —NR′R″,

g) —C(═NR′)—NR″R′″,

h) —NHC(═NR′)—NR″R′″,

i) —NHCOR′, and

j) a hydroxy group,

Here, R′, R″, and R′″ in b) and f) to i) each independently represent ahydrogen atom or a C1 to C6 alkyl group, and are preferably eachindependently a hydrogen atom or a C1 to C3 alkyl group.

a) to j) are preferably as follows:

a) a C1 to C3 alkoxy group optionally substituted with one to threehalogen atoms, more preferably a methoxy group, an ethoxy group, ann-propoxy group, an i-propoxy group, or a trifluoromethoxy, even morepreferably a methoxy group, an ethoxy group, or a trifluoromethoxygroup, and most preferably a methoxy group;b) a C1 to C3 alkyl group optionally substituted with one to threehalogen atoms, a hydroxy group, —OCOR′, —C(═NR′)—NR″R′″, or—NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably a C1 to C3alkyl group optionally substituted with any selected from one to threehalogen atoms, a hydroxy group, —OCOR′, —C(═NR′)—NR″R′″, and—NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a methyl group, even more preferably a methyl group, anethyl group, a n-propyl group, an i-propyl group, a fluoromethyl group,a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group,—CH₂OCOMe, —CH₂—NHC(═NH)—NH₂, or —CH₂—NHC(═NMe)-NH₂;c) a halogen atom, preferably a fluorine atom or a chlorine atom;d) a C3 to C5 cycloalkoxy group, more preferably a cyclopropoxy group;e) a C1 to C3 alkylthio group, more preferably a methylthio group or anethylthio group;f) —NR′R″, wherein R′ and R″ are each independently a hydrogen atom or aC1 to C3 alkyl group, more preferably —NH₂, —NHMe, —NMe₂, —NHEt, or-NEt₂;g) —C(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably —C(═NH)—NH₂ or—C(═NMe)-NH₂;h) —NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably —NHC(═NH)—NH₂or —NHC(═NMe)-NH₂;i) —NHCOR′, wherein R′ is a hydrogen atom or a C1 to C3 alkyl group,more preferably —NHCOMe or -NHCOEt; andj) a hydroxy group.

The aryl group (preferably, a phenyl group) or heteroaryl group(preferably, a pyridyl group) for R⁵ may have at least one substituentat any position. If a plurality of substituents is present, thesubstituents may be the same or different.

If R⁵ is an aryl group, each substituent is preferably a), b), d), g),h), or j), and more preferably a), b), d), or j).

If R⁵ is a phenyl group, R⁵ may have a substituent at any position andmay have a plurality of substituents, and preferably one or twosubstituents are present at the 3-position and/or the 4-position, andmore preferably one substituent is present at the 4-position.

If R⁵ is a naphthyl group, R⁵ may have a substituent at any position andmay have a plurality of substituents, and preferably one substituent ispresent at the 6-position.

If R⁵ is a phenyl group, R⁵ is more preferably a phenyl group, a4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group,a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, or a4-carbamimidamidomethyl-phenyl group, and even more preferably a phenylgroup, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, ora 4-trifluoromethylphenyl group.

If R⁵ is a naphthyl group, R⁵ is more preferably a naphthyl group or a6-methoxy-2-naphthyl group.

The most preferred is a 4-methoxyphenyl group.

If R⁵ is a heteroaryl group, each substituent is preferably a), b), d),g), h), or j), and more preferably a) or b).

If R⁵ is a heteroaryl group, R⁵ may have at least one substituent at anyposition. If R⁵ is a 3-pyridyl group, its substituent(s) is preferablypresent at the 6-position and/or the 5-position. If R⁵ is 2-pyridyl, itssubstituent(s) is preferably present at the 5-position and/or the4-position, or at the 5-position and/or the 6-position. If R⁵ is4-pyridyl, its substituent(s) is preferably present at the 2-positionand/or the 6-position.

If R⁵ is a heteroaryl group, R⁵ may have a plurality of substituents,and preferably has one or two substituents, and preferably has onesubstituent.

If R⁵ is a pyridyl group, R⁵ is preferably a 6-methoxy-3-pyridyl groupor a 6-methyl-3-pyridyl group.

If R⁵ is a 3-thienyl group or a 6-quinoxalyl group, R⁵ is preferablyunsubstituted.

“C1 to C6 alkyl group” in “C1 to C6 alkyl group optionally having one ormore substituents selected from group 2” for R⁵ is preferably a C1 to C3alkyl group, and more preferably a methyl group or an ethyl group.

The substituents in “C1 to C6 alkyl group optionally having one or moresubstituents selected from group 2” for R⁵ are each a halogen atom, ahydroxy group, or a C1 to C6 alkoxy group (preferably, a C1 to C3 alkoxygroup), preferably a hydroxy group, a methoxy group, or an ethoxy group,and more preferably a hydroxy group.

(ii) If R³ represents a hydrogen atom, R⁴ and R⁵ are combined, togetherwith the carbon atom to which R⁴ and R⁵ are bound, to form a three- tofive-membered saturated hydrocarbon ring or three- to five-memberedsaturated heterocycle, or CH₂═ as shown in the following:

The three- to five-membered saturated hydrocarbon ring may besubstituted with one to four halogen atoms, and may be preferablysubstituted with one or two fluorine atoms.

R⁴ and R⁵ are preferably combined to form a three- to five-memberedsaturated hydrocarbon ring or CH₂═, more preferably to formcyclopropane, cyclobutane, or CH₂═(exomethylene group), and even morepreferably to form cyclopropane.

If R⁴ and R⁵ are combined to form a three- to five-membered saturatedhydrocarbon ring or three- to five-membered saturated heterocycle, thethree- to five-membered saturated hydrocarbon ring or three- tofive-membered saturated heterocycle is preferably the same as A. Morepreferably, A is a three- to five-membered saturated hydrocarbon ringand R⁴ and R⁵ are combined to form a three- to five-membered saturatedhydrocarbon ring, and even more preferably A is a cyclopropane ring andR⁴ and R⁵ are combined to form a cyclopropane ring.

(iii) R³, R⁴, and R⁵ are combined, together with the carbon atom towhich R³ is bound and the carbon atom to which R⁴ and R⁵ are bound, toform a benzene ring or six-membered heterocycle optionally having one ormore substituents selected from group 3.

The following formula shows the case in which R³ and R⁴ are combined toform a benzene ring optionally having one or more substituents:

The benzene ring or heterocycle may have at least one substituent at anyposition. If a plurality of substituents is present, the substituentsmay be the same or different.

Each substituent of the benzene ring or the heterocycle is a halogenatom, a C1 to C6 alkyl group optionally substituted with one to threehalogen atoms, or a C1 to C6 alkoxy group, preferably a halogen atom, aC1 to C3 alkyl group optionally substituted with one to three halogenatoms, or a C1 to C3 alkoxy, and more preferably a halogen atom, amethyl group, or a methoxy group.

“Benzene ring or six-membered heterocycle optionally having one or moresubstituents” is preferably an unsubstituted benzene ring.

R³, R⁴ and R⁵ most preferably satisfy the above (i).

R⁶ and R⁷ each represent a hydrogen atom, or R⁶ and R⁷ are combined torepresent an imine bond (C═N).

R⁸ is a hydroxy group or a C1 to C3 alkoxy group, preferably a hydroxygroup or a methoxy group, and more preferably a hydroxy group. R⁸ may bea hydrogensulfite adduct (OSO₃M, wherein M is a metal cation).

Since R¹ bonds to an asymmetric carbon atom, a steric configurationrepresented by partial structure (Va) or (Vb) below is provided. Eachwavy line represents bonding to Y in general formula (V), and eachasterisk represents bonding to L.

X and Y are each independently an oxygen atom, a nitrogen atom, or asulfur atom, and preferably an oxygen atom.

Drug D of the present invention is preferably any one compound selectedfrom the following group:

<Linker Structure>

The linker structure to bond the antitumor drug to the antibody in theantibody-drug conjugate of the present invention will be described.

Linker L is represented by the following formula:-Lb-La-Lp-NH—B—CH₂—O(C═O)—*

The asterisk represents bonding to the nitrogen atom at theN10′-position of drug D, Lb represents a spacer which connects La to aglycan or remodeled glycan of Ab, or a spacer which connects La to aside chain of an amino acid residue (e.g., cysteine, or lysine) ofantibody Ab.

B represents a phenyl group or a heteroaryl group, and is preferably a1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a1,4-phenyl group.

Lp represents a linker consisting of an amino acid sequence cleavable invivo or in a target cell. Lp is, for example, cleaved by the action ofan enzyme such as esterase and peptidase.

Lp is a peptide residue composed of two to seven (preferably, two tofour) amino acids. That is, Lp is composed of an oligopeptide residue inwhich two to seven amino acids are connected via peptide bonding.

Lp is bound at the N terminal to a carbonyl group of La in Lb-La—, andforms at the C terminal an amide bond with the amino group (—NH—) of thepart —NH—B—CH₂—O(C═O)— of the linker. The bond between the C terminal ofLp and —NH— is cleaved by the enzyme such as esterase.

The amino acids constituting Lp are not limited to particular aminoacids, and, for example are L- or D-amino acids, and preferably L-aminoacids. The amino acids may be not only α-amino acids, but may include anamino acid with structure, for example, of β-alanine, ε-aminocaproicacid, or γ-aminobutyric acid, and may further include a non-naturalamino acid such as an N-methylated amino acid.

The amino acid sequence of Lp is not limited to a particular amino acidsequence, and examples of amino acids that constitute Lp may include,but are not limited to, glycine (Gly; G), valine (Val; V), alanine (Ala;A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I),proline (Pro; P), citrulline (Cit), leucine (Leu; L), serine (Ser; S),lysine (Lys; K), and aspartic acid (Asp; D). Preferred among them areglycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline(Cit).

Any of these amino acids may appear multiple times, and Lp has an aminoacid sequence including arbitrarily selected amino acids. Drug releasepattern may be controlled via amino acid type.

Specific examples of linker Lp may include, but are not limited to,-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,-GGPL-, -EGGVA, -PI-, -GGF-, DGGF-, (D-)D-GGF-, -EGGF-, -SGGF-, -KGGF-,-DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-.

Here, “(D-)V” indicates D-valine, “(D)-P” indicates D-proline, and“(D-)D” indicates D-aspartic acid.

Linker Lp is preferably any of the following:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,and -GGPL-.

Linker Lp is more preferably any of the following:

-GGVA-, -GGVCit-, and -VA-.

Lb represents: i) a spacer which connects La to a glycan or remodeledglycan of Ab; or ii) a spacer which connects La to a side chain of anamino acid residue (e.g., cysteine, or lysine) of antibody Ab.

If Lb is i), Lb represents any one selected from the following group:

—C(═O)—(CH₂CH₂)n²-C(═O)—, —C(═O)—(CH₂CH₂)n²-C(═O)—NH—(CH₂CH₂)n³-C(═O)—,

—C(═O)—(CH₂CH₂)n²-C(═O)—NH—(CH₂CH₂O)n³-CH₂—C(═O)—,

—C(═O)—(CH₂CH₂)n²-NH—C(═O)—(CH₂CH₂O)n³-CH₂CH₂—C(═O)—, —(CH₂)n⁴-O—C(═O)—

wherein,

n² represents an integer of 1 to 3 (preferably, 1 or 2), n³ representsan integer of 1 to 5 (preferably, an integer of 2 to 4, more preferably,2 or 4), and n⁴ represents an integer of 0 to 2 (preferably, 0 or 1).

If Lb is i), La preferably represents any one selected from thefollowing group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—,

—CH₂—OC(═O)—, and —OC(═O)—, and

La is more preferably —C(═O)—CH₂CH₂—C(═O)— or —C(═O)—(CH₂CH₂)₂—C(═O)—.

Spacer Lb is not limited to a particular spacer, and examples thereofmay include, but are not limited to, a spacer represented by thefollowing formulas.

In the structural formulas for Lb shown above, each asterisk representsbonding to —(C═O) or —(CH₂)n⁴ at the left end of La, and each wavy linerepresents bonding to a glycan or remodeled glycan of Ab.

In each structural formula for Lb (Lb-1, Lb-2, or Lb-3) shown above, thetriazole ring site formed through click reaction of an azide group andDBCO provides structures of geometric isomers, and molecules of Lb existas any one of the two structures or as a mixture of both of them. Thereexist m¹″-L-D″ moieties per molecule of the antibody-drug conjugate ofthe present invention, and either one of the two structures exist orboth of them coexist as Lb (Lb-1, Lb-2, or Lb-3) in L of each of them¹″-L-D″ moieties.

If Lb is i), L is preferably represented by -Lb-La-Lp-NH—B—CH₂—O(C═O)—*,wherein

B is a 1,4-phenyl group,

Lp represents any one selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,and -GGPL-,

La represents any one selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, —OC(═O)—

and

Lb represents any of the structural formulas above for Lb.

If Lb is i), L is more preferably any one selected from the followinggroup:

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GG-(D-)VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGPI-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGFG-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVK-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGPL-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z²—OC(═O)-GGVA-NH—B—CH₂—OC(═O)—, —Z³—CH₂—OC(═O)-GGVA-NH—B—CH₂—OC(═O)—

wherein

Z¹ represents the following structural formula as described for Lb:

Z² represents the following structural formula as described for Lb:

Z³ represents the following structural formula as described for Lb:

and B is a 1,4-phenyl group.

L is most preferably any of the following:

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

—Z¹—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,

and—Z¹—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—,wherein

B is a 1,4-phenyl group, and Z¹ represents the following structuralformula as described for Lb:

If Lb is ii) and the amino acid residue is a cysteine residue, thespacer Lb is not limited to a particular spacer, and examples thereofmay include, but are not limited to, -(succinimid-3-yl-N)—.

“-(succinimid-3-yl-N)—” has a structure represented by the followingstructure:

In the structural formula shown above, the asterisk represents bondingto La. The wavy line represents bonding to the thiol group of a cysteineresidue of the antibody via a thiol bond, and the bonding may besite-specific cysteine conjugation (RSC Adv., 2017, 7, 24828-24832,etc.).

If Lb is ii), L is represented by -Lb-La-Lp-NH—B—CH₂—O(C═O)—*, wherein

B is a 1,4-phenyl group;

Lp represents any one of -GGVA-, -GG-(D-)VA-, -VA-, and -GGFG-;

La represents —(CH₂)n⁹-C(═O)— or—(CH₂CH₂)n¹⁰-C(═O)—NH—(CH₂CH₂O)n¹¹-CH₂CH₂—C(═O)—, wherein

n⁹ represents an integer of 2 to 7 (preferably, an integer of 2 to 5,more preferably 2, or 5), n¹⁰ represents an integer of 1 to 3(preferably, 1), and n¹¹ represents an integer of 6 to 10 (preferably,8); and

Lb represents -(succinimid-3-yl-N)—.

If Lb is ii), L is preferably any of the following:

-(Succinimid-3-yl-N)—(CH₂)₅—C(═O)—VA-NH—B—CH₂—OC(═O)—,

-(Succinimid-3-yl-N)—(CH₂)₅—C(═O)-GGVA-NH—B—CH₂—OC(═O)—, or,

-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—VA-NH—B—CH₂—OC(═O)—

wherein B is a 1,4-phenyl group.

The antibody-drug conjugate of the present invention is inferred toexhibit antitumor activity through a process in which most molecules ofthe antibody-drug conjugate migrate into tumor cells, and a linkerportion (e.g., Lp) is then cleaved by an enzyme or the like to activatethe antibody-drug conjugate, which releases the portion of drug D(hereinafter, referred to as a free drug (described later)).

Therefore, it is preferable that the antibody-drug conjugate of thepresent invention is stable outside of tumor cells.

<Free Drug and Production Intermediate>

The intermediate and free drug of the antibody-drug conjugate of thepresent invention is represented by the following formula:

This will be described in the following.

The free drug of the present invention is generated through a process inwhich the antibody-drug conjugate migrates into tumor cells and theportion of linker L in the antibody-drug conjugate is then cleaved.Examples of the free drug may include, but are not limited to, drugs 1to 16 in Examples 45 to 54 and 150 to 152.

The antibody-drug conjugate of the present invention is produced byusing the production intermediate.

The free drug for the antibody-drug conjugate of the present inventioncorresponds to the case in which (a) R¹⁶ and R¹⁷ are combined to form animine bond (N═C).

The production intermediate for the antibody-drug conjugate of thepresent invention corresponds to the case in which (b) R¹⁶ isrepresented by J-La′-Lp′-NH—B′—CH₂—O (C═O)—*.

Accordingly, 1 and n¹, E and A, R⁹ and R¹, R¹⁰ and R², R¹¹ and R³, R¹²and R⁴, R¹³ and R⁵, R¹⁴ and R⁶, R¹⁵ and R⁷, V and X, W and Y, group 7and group 1, group 8 and group 2, group 9 and group 3, group 10 andgroup 4, group 11 and group 5, and group 12 and group 6 in the formulasare respectively synonymous.

l represents an integer of 2 to 8, and is preferably an integer of 2 to6, and more preferably an integer of 3 to 5.

The alkyl chain with 1 being an integer of 2 to 8, preferably an integerof 2 to 6, and more preferably an integer of 3 to 5, may include adouble bond.

E represents a spiro-bonded three- to five-membered saturatedhydrocarbon ring or a three- to five-membered saturated heterocycle, andis preferably a three- to five-membered saturated hydrocarbon ring(cyclopropane, cyclobutane, or cyclopentane), more preferablycyclopropane or cyclobutane, and most preferably cyclopropane.

The spiro-bonded three- to five-membered saturated hydrocarbon ring maybe substituted with one to four halogen atoms, and may be preferablysubstituted with one or two fluorine atoms (e.g.,2,2-difluorocyclopropane).

R⁹ and R¹⁰ each independently represent a C1 to C6 alkoxy group, a C1 toC6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1 toC6 alkylthio group, a halogen atom, or —NR′R″, and are each preferably aC1 to C6 alkoxy group, a C1 to C6 alkyl group, or a hydroxy group, morepreferably a C1 to C3 alkoxy group, and most preferably a methoxy group.

R¹¹, R¹², and R¹³ are as described in any of the following (i) to (iii).

(i) If R¹¹ and R¹² are combined together with the carbon atoms to whichR³ and R⁴ are bound to form a double bond, R¹³ represents an aryl groupor heteroaryl group optionally having one or more substituents selectedfrom group 7 or a C1 to C6 alkyl group optionally having one or moresubstituents selected from group 8, and is preferably an aryl groupoptionally having one or more substituents selected from group 7.

“Aryl group” in “aryl group or heteroaryl group optionally having one ormore substituents selected from group 7” for R¹³ is preferably a phenylgroup or a naphthyl group, and more preferably a phenyl group.

“Heteroaryl group” in “aryl group or heteroaryl group optionally havingone or more substituents selected from group 7” for R¹³ is preferably athienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, aquinoxalyl group, or a benzothiophenyl group, more preferably a2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridylgroup, or a 4-pyridyl group, and even more preferably a 3-pyridyl groupor a 3-thienyl group.

Examples of substituents of the aryl group or heteroaryl group for R¹³may include, but are not limited to, the following a) to j):

a) a C1 to C6 alkoxy group optionally substituted with one to threehalogen atoms,

b) a C1 to C6 alkyl group optionally substituted with any one selectedfrom one to three halogen atoms, a hydroxy group, —OCOR′, —NR′R″,—C(═NR′)—NR″R′″, and —NHC(═NR′)—NR″R′″,

c) a halogen atom,

d) a C3 to C5 cycloalkoxy group,

e) a C1 to C6 alkylthio group,

f) —NR′R″,

g) —C(═NR′)—NR″R′″,

h) —NHC(═NR′)—NR″R′″,

i) —NHCOR′, and

j) a hydroxy group,

Here, R′, R″, and R′″ in b) and f) to i) each independently represent ahydrogen atom or a C1 to C6 alkyl group, and are preferably eachindependently a hydrogen atom or a C1 to C3 alkyl group.

a) to j) are preferably as follows:

a) a C1 to C3 alkoxy group optionally substituted with one to threehalogen atoms, more preferably a methoxy group, an ethoxy group, an-propoxy group, an i-propoxy group, or a trifluoromethoxy group, evenmore preferably a methoxy group, an ethoxy group, or a trifluoromethoxygroup, most preferably a methoxy group;b) a C1 to C3 alkyl group optionally substituted with any selected fromone to three halogen atoms, a hydroxy group, —OCOR′, —C(═NR′)—NR″R′″,and —NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably a C1 to C3alkyl group optionally substituted with any selected from one to threehalogen atoms, a hydroxy group, —OCOR′, —C(═NR′)—NR″R′″, and—NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a methyl group, even more preferably a methyl group, anethyl group, a n-propyl group, an i-propyl group, a fluoromethyl group,a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group,—CH₂OCOMe, —CH₂—NHC(═NH)—NH₂, or —CH₂—NHC(═NMe)-NH₂;c) a halogen atom, preferably a fluorine atom or a chlorine atom;d) a C3 to C5 cycloalkoxy group, more preferably a cyclopropoxy group;e) a C1 to C3 alkylthio group, more preferably a methylthio group or anethylthio group;f) —NR′R″, wherein R′ and R″ are each independently a hydrogen atom or aC1 to C3 alkyl group, more preferably —NH₂, —NHMe, —NMe₂, —NHEt, or-NEt₂;g) —C(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably —C(═NH)—NH₂ or—C(═NMe)-NH₂;h) —NHC(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently ahydrogen atom or a C1 to C3 alkyl group, more preferably —NHC(═NH)—NH₂or —NHC(═NMe)-NH₂;i) —NHCOR′, wherein R′ is a hydrogen atom or a C1 to C3 alkyl group,more preferably —NHCOMe or -NHCOEt; andj) a hydroxy group.

The aryl group (preferably, a phenyl group) or heteroaryl group(preferably, a pyridyl group) for R¹³ may have at least one substituentat any position. If a plurality of substituents is present, thesubstituents may be the same or different.

If R¹³ is an aryl group, each substituent is preferably a), b), d), g),h), or j), and more preferably a), b), d), or j).

If R³ is a phenyl group, R¹³ may have a substituent at any position andmay have a plurality of substituents, and preferably one or twosubstituents are present at the 3-position and/or the 4-position, andmore preferably one substituent is present at the 4-position.

If R⁵ is a naphthyl group, R⁵ may have a substituent at any position andmay have a plurality of substituents, and preferably one substituent ispresent at the 6-position.

If R³ is a phenyl group, R¹³ is more preferably a phenyl group, a4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group,a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, or a4-carbamimidamidomethyl-phenyl group, and even more preferably a phenylgroup, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, ora 4-trifluoromethylphenyl group.

If R¹³ is a naphthyl group, R¹³ is more preferably a naphthyl group or a6-methoxy-2-naphthyl group.

The most preferred is a 4-methoxyphenyl group.

If R¹³ is a heteroaryl group, each substituent is preferably a), b), d),g), h), or j), and more preferably a) or b).

If R¹³ is a heteroaryl group, R¹³ may have at least one substituent atany position. If R¹³ is a 3-pyridyl group, its substituent(s) ispreferably present at the 6-position and/or the 5-position. If R¹³ is2-pyridyl, its substituent(s) is preferably present at the 5-positionand/or the 4-position or at the 5-position and/or the 6-position. If R³is 4-pyridyl, its substituent is preferably present at the 2-positionand/or the 6-position.

If R³ is a heteroaryl group, R¹³ may have a plurality of substituents,and preferably has one or two substituents, and preferably has onesubstituent.

If R¹³ is a pyridyl group, R¹³ is preferably a 6-methoxy-3-pyridyl groupor a 6-methyl-3-pyridyl group.

If R³ is a 3-thienyl group or a 6-quinoxalyl group, R¹³ is preferablyunsubstituted.

“C1 to C6 alkyl group” in “C1 to C6 alkyl group optionally having one ormore substituents selected from group 8” for R¹³ is preferably a C1 toC3 alkyl group, and more preferably a methyl group or an ethyl group.

The substituents in “C1 to C6 alkyl group optionally having one or moresubstituents selected from group 8” for R¹³ are each a halogen atom, ahydroxy group, or a C1 to C6 alkoxy group (preferably, a C1 to C3 alkoxygroup), preferably a hydroxy group, a methoxy group, or an ethoxy group,and more preferably a hydroxy group.

(ii) If R¹¹ represents a hydrogen atom, R¹² and R¹³ are combined,together with the carbon atom to which R¹² and R¹³ are bound, to form athree- to five-membered saturated hydrocarbon ring or a three- tofive-membered saturated heterocycle, or CH₂═.

The three- to five-membered saturated hydrocarbon ring may besubstituted with one to four halogen atoms, and may be preferablysubstituted with one or two fluorine atoms.

R¹² and R¹³ are preferably combined to form a three- to five-memberedsaturated hydrocarbon ring or CH₂═, more preferably to formcyclopropane, cyclobutane, or CH₂═(exomethylene group), and even morepreferably to form cyclopropane.

If R¹² and R¹³ are combined to form a three- to five-membered saturatedhydrocarbon ring or a three- to five-membered saturated heterocycle, thethree- to five-membered saturated hydrocarbon ring or a three- tofive-membered saturated heterocycle is preferably the same as E. Morepreferably, E is a three- to five-membered saturated hydrocarbon ringand R¹² and R¹³ are combined to form a three- to five-membered saturatedhydrocarbon ring, and even more preferably E is a cyclopropane ring andR¹² and R¹³ are combined to form a cyclopropane ring.

(iii) R¹¹, R¹², and R¹³ are combined, together with the carbon atom towhich R¹¹ is bound and the carbon atom to which R¹² and R¹³ are bound,to form a benzene ring or six-membered heterocycle optionally having oneor more substituents selected from group 9.

The benzene ring or heterocycle may have at least one substituent at anyposition. If a plurality of substituents is present, the substituentsmay be the same or different.

Each substituent of the benzene ring or heterocycle is a halogen atom, aC1 to C6 alkyl group optionally substituted with one to three halogenatoms, or a C1 to C6 alkoxy group, preferably a halogen atom, a C1 to C3alkyl group optionally substituted with one to three halogen atoms, or aC1 to C3 alkoxy, and more preferably a halogen atom, a methyl group, ora methoxy group.

“Benzene ring or six-membered heterocycle optionally having one or moresubstituents” is preferably an unsubstituted benzene ring.

R¹¹, R¹² and R¹³ most preferably satisfy the above (i).

R¹⁴ and R¹⁵ each represent a hydrogen atom, or R¹⁴ and R¹¹ are combinedto represent an imine bond (C═N).

V and W are each independently an oxygen atom, a nitrogen atom, or asulfur atom, and preferably an oxygen atom.

R¹⁶ and R¹⁷ are such that:

(a) R¹⁶ and R¹⁷ are combined to form an imine bond (N═C); or

(b) R¹⁶ represents J-La′-Lp′-NH—B′—CH₂—O(C═O)—* and R¹⁷ represents ahydroxy group or a C1 to C3 alkoxy group.

In the case of (b) R¹⁶ is J-La′-Lp′-NH—B′—CH₂—O(C═O)—*, the asterisk inthe formula represents bonding to the N10′-position of thepyrrolobenzodiazepine ring represented by the above formula.

B′ represents a phenyl group or a heteroaryl group, and is preferably a1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a1,4-phenyl group.

Lp′ represents a linker consisting of an amino acid sequence cleavablein vivo or in a target cell. Lp is, for example, cleaved by the actionof an enzyme such as esterase and peptidase.

Specific examples of linker Lp′ may include, but are not limited to,-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,-GGPL-, -EGGVA, -PI-, -GGF-, DGGF-, (D-)D-GGF-, -EGGF-, -SGGF-, -KGGF-,-DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-.

Here, “(D-)V” indicates D-valine, “(D)-P” indicates D-proline, and“(D-)D” indicates D-aspartic acid.

Linker Lp′ is preferably as follows:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, -GG(D-)PI-,or -GGPL-.

More preferred examples are -GGVA-, -GGVCit-, and -VA-.

La′ represents any one selected from the following group:—C(═O)—(CH₂CH₂)n⁶-C(═O)—, —C(═O)—(CH₂CH₂)n⁶-C(═O)—NH—(CH₂CH₂)n⁷-C(═O)—,—C(═O)—(CH₂CH₂)n⁶-C(═O)—NH—(CH₂CH₂O)n⁷-CH₂—C(═O)—,—C(═O)—(CH₂CH₂)n⁶-NH—C(═O)—(CH₂CH₂O)n⁷-CH₂CH₂—C(═O)—, —(CH₂)n⁸-O—C(═O)—,—(CH₂)n¹²-C(═O)—, and, —(CH₂CH₂)n¹³-C(═O)—NH—(CH₂CH₂O)n¹⁴-CH₂CH₂—C(═O)—

In the formulas, n⁶ represents an integer of 1 to 3 (preferably, 1 or2), n⁷ represents an integer of 1 to 5 (preferably, an integer of 2 to4, more preferably, 2 or 4), n⁸ represents an integer of 0 to 2(preferably, 0 or 1), n¹² represents an integer of 2 to 7 (preferably,an integer of 2 to 5, more preferably, 2 or 5), n¹³ represents aninteger of 1 to 3 (preferably, 1), and n¹⁴ represents an integer of 6 to10 (preferably, 8).

La′ preferably represents any one selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —CH₂—OC(═O)—, —OC(═O)—,

—(CH₂)₂—C(═O)—, —(CH₂)₅—C(═O)—, and—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—.

La′ is more preferably —C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—, or—(CH₂)₅—C(═O)—.

J is not limited to a particular structure and may be any cyclicstructure including an alkyne structure that reacts with an azide groupto form a 1,2,3-triazole ring, and examples thereof may include, but arenot limited to, compounds represented by the following formulas:

In the structural formulas for J shown above, each asterisk representsbonding to —(C═O) or —(CH₂)n⁸ at the left end of La′.

Alternatively, J may be a compound that bonds to a side chain of anamino acid residue (e.g., cysteine, or lysine) of antibody Ab, or ahalogen atom, and examples of J may include, but are not limited to, amaleimidyl group represented by the following formula:

In the maleimidyl group shown above, the asterisk represents bonding to—(CH₂)n¹² or —(CH₂CH₂)n¹³ at the left end of La′.

R¹⁶ is preferably represented by J-La′-Lp′-NH—B′—CH₂—O(C═O)—*, wherein

B′ is a 1,4-phenyl group;

Lp′ represents any one selected from the following group:

-GGVA-, -GG-(D-)VA-, -VA-, -GGFG-, -GGPI-, -GGVCit-, -GGVK-, GG(D-)PI-,and -GGPL-;

La′ represents any one selected from the following group:

—C(═O)—CH₂CH₂—C(═O)—, —C(═O)—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—,

—C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—,

—C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—, —OC(═O)—, —CH₂—OC(═O)—,

—(CH₂)₅—C(═O)—, and —CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—; and

J represents any of the structural formulas:

wherein, in the structural formulas for J,

each asterisk represents bonding to La′.

R¹⁶ is more preferably any one selected from the following group:

J¹-C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GG-(D-)VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGPI-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGFG-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVK-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGPL-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J²-OC(═O)-GGVA-NH—B′—CH₂—OC(═O)—, J³-CH₂—OC(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)—VA-NH—B′—CH₂—OC(═O)—, and

J⁴-CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₈—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—

wherein J¹, J², J³, and J⁴ represent structural formulas represented bythe following:

wherein, in the structural formulas for J¹, J², J³ and J⁴,

each asterisk represents bonding to a group neighboring to J¹, J², J³,or J⁴, and

B′ is a 1,4-phenyl group.

R¹⁶ is most preferably any of the following:

J¹-C(═O)—CH₂CH₂—C(═O)-GGVA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)-GGVCit-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂)₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—C(═O)—NH—(CH₂CH₂O)₂—CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J¹-C(═O)—CH₂CH₂—NH—C(═O)—(CH₂CH₂O)₄—CH₂CH₂—C(═O)—VA-NH—B′—CH₂—OC(═O)—,

J⁴-(CH₂)₅—C(═O)—VA-NH—B′—CH₂—OC(═O)—

wherein

B′ is a 1,4-phenyl group, and

J¹ and J⁴ are represented by the following structural formulas for J:

wherein, in the structural formulas for J¹ and J⁴,

each asterisk represents bonding to a group neighboring to J¹ or J⁴.

R¹⁷ is a hydroxy group or a C1 to C3 alkoxy group, and preferably ahydroxy group or a methoxy group.

R⁷ may be hydrogensulfite adduct (OSO₃M, wherein M is a metal cation).

Since R¹⁷ bonds to an asymmetric carbon atom, a steric configurationrepresented by partial structure (VIa) or (VIb) below is provided. Eachwavy line represents bonding to W in the intermediate and free drugrepresented by general formula (VI).

The free drug is preferably one compound selected from the followinggroup:

The free drug is in some cases released in tumor cells with a part oflinker L bonded, but is a superior drug that exerts superior antitumoreffect even in such state. The free drug, after migrating to tumorcells, is in some cases further oxidized to cause dehydrogenation of R¹⁶and R¹⁷, but exerts superior antitumor effect even in such state.

The production intermediate is preferably one compound selected from thefollowing group:

The production intermediate is preferably one compound selected from thefollowing group:

<Antibody>

In the present invention, “cancer” and “tumor” are used for the samemeaning.

In the present invention, a “gene” refers to nucleotides or a nucleotidesequence including a nucleotide sequence encoding amino acids of proteinor a complementary strand thereof. The meaning of a “gene” encompasses,for example, a polynucleotide, an oligonucleotide, DNA, mRNA, cDNA, andRNA as a nucleotide sequence including a nucleotide sequence encodingamino acids of protein or a complementary strand thereof. Examples ofthe “CLDN6 gene” of the present invention include DNA, mRNA, cDNA, andcRNA including a nucleotide sequence encoding the amino acid sequence ofCLDN6 protein.

In the present invention, “nucleotides”, “polynucleotide”, and“nucleotide sequence” have the same meaning as that of “nucleic acids”,and the meaning of “nucleotides” and “nucleotide sequence” encompasses,for example, DNA, RNA, a probe, an oligonucleotide, a polynucleotide,and a primer.

In the present invention, “polypeptide”, “peptide”, and “protein” areused interchangeably.

In the present invention, “CLDN6” is used for the same meaning as CLDN6protein.

In the present invention, “cells” include cells in an animal individualand cultured cells.

In the present invention, “cellular cytotoxic activity” refers tocausing pathological change to cells in any way, which includes causing,not only direct traumas, but also all types of damage in the structureand function of cells such as cleavage of DNA, formation of a nucleotidedimer, cleavage of a chromosome, damage of the mitotic apparatus, andlowered activity of various enzymes.

In the present invention, a “functional fragment of an antibody” is alsoreferred to as an “antigen-binding fragment of an antibody”, and means apartial fragment of an antibody with binding activity to an antigen, andexamples thereof may include, but not limited to, Fab, F(ab′)2, Fv,scFv, diabodies, linear antibodies, and multispecific antibodies formedfrom antibody fragments. In addition, the meaning of an antigen-bindingfragment of an antibody encompasses Fab′, a monovalent fragment of avariable region of an antibody obtained by treating F(ab′)2 underreducing conditions. However, there is no limitation to those moleculesas long as the molecules have binding ability to an antigen. Thoseantigen-binding fragments include not only those obtained by treating afull-length molecule of an antibody protein with an appropriate enzyme,but also protein produced in an appropriate host cell by using agenetically engineered antibody gene.

The functional fragment of the present invention includes a functionalfragment that has well conserved asparagine (Asn297) to be modified withan N-linked glycan in the IgG heavy chain Fc region and amino acidsaround Asn297, while retains binding activity to an antigen.

In the present invention, an “epitope” refers to a partial peptide orpartial three-dimensional structure of an antigen to which a particularantibody (e.g., an anti-CLDN6 antibody) binds (a partial peptide orpartial three-dimensional structure of CLDN6). An epitope as such apartial peptide (e.g., a partial peptide of CLDN6) can be determined byusing any method well known to those skilled in the art, such asimmunoassay.

A “CDR” in the present invention refers to a complementarity determiningregion. It is known that each of heavy chains and light chains of anantibody molecule have three CDRs. CDRs, which are also called ahypervariable region, are located in variable regions of heavy chainsand light chains of an antibody and is a site with particularly highvariation of the primary structure. Three CDRs are separately located inthe primary structure of the polypeptide chain of each of heavy chainsand light chains. Regarding CDRs of antibodies, herein, CDRs of a heavychain refer to CDRH1, CDRH2, and CDRH3 from the amino terminus of theheavy chain amino acid sequence, and CDRs of a light chain refer toCDRL1, CDRL2, and CDRL3 from the amino terminus of the light chain aminoacid sequence. These sites are located in the proximity of each other inthe three-dimensional structure, determining specificity to an antibodyto bind.

In the present invention, “hybridize under stringent conditions” refersto hybridization in the commercially available hybridization solutionExpressHyb Hybridization Solution (Clontech) at 68° C., or hybridizationusing a filter with DNA fixed thereto in the presence of 0.7 to 1.0 MNaCl at 68° C. and washing at 68° C. with 0.1 to 2×SSC solution (1×SSCsolution contains 150 mM NaCl and 15 mM sodium citrate), orhybridization under conditions equivalent thereto.

In the present invention, “one to several” refers to 1 to 10, one tonine, one to eight, one to seven, one to six, one to five, one to four,one to three, or one or two.

In the present invention, an antibody capable of recognizing or bindingto CLDN6 and that capable of recognizing or binding to CLDN6 and CLDN9are occasionally called as an “anti-CLDN6 antibody” and an“anti-CLDN6/CLDN9 antibody”, respectively. Such antibodies includechimeric antibodies, humanized antibodies, and human antibodies. Anantibody capable of recognizing or binding to CLDN6 and CLDN9 isoccasionally called as an “anti-CLDN6 antibody”.

The antibody to be used for the antibody-drug conjugate of the presentinvention refers to immunoglobulin, and is a molecule including anantigen-binding site which immunospecifically binds to an antigen. Theantibody of the present invention may be of any class of IgG, IgE, IgM,IgD, IgA, and IgY, and preferred is IgG. The subclass may be any ofIgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and preferred are IgG1, IgG2,and IgG4. If IgG1 or IgG4 is used, the effector function may be adjustedby substituting some of amino acid residues in the constant region (seeWO 88/07089, WO 94/28027, WO 94/29351).

The antibody may be derived from any species, which preferably include,but not limited to, a human, a rat, a mouse, and a rabbit. If theantibody is derived from species other than human species, it ispreferably chimerized or humanized using a well known technique. Theantibody of the present invention may be a polyclonal antibody or amonoclonal antibody, and is preferably a monoclonal antibody. Examplesof monoclonal antibodies may include, but not limited to, monoclonalantibodies derived from non-human animals such as rat antibodies, mouseantibodies, and rabbit antibodies; chimeric antibodies; humanizedantibodies; human antibodies; functional fragments of them; and modifiedvariants of them.

The antibody of the present invention is preferably an antibody capableof targeting a tumor cell. Specifically, the antibody, to which a drughaving antitumor activity is conjugated via a linker, preferably has oneor more properties of recognizing a tumor cell, binding to a tumor cell,being incorporated and internalizing in a tumor cell, and damaging atumor cell.

The binding activity of the antibody against tumor cells can beconfirmed using flow cytometry. The incorporation of the antibody intotumor cells can be confirmed using (1) an assay of visualizing anantibody incorporated in cells under a fluorescence microscope using asecondary antibody (fluorescently labeled) binding to the therapeuticantibody (Cell Death and Differentiation (2008) 15, 751-761), (2) anassay of measuring a fluorescence intensity incorporated in cells usinga secondary antibody (fluorescently labeled) binding to the therapeuticantibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December2004), or (3) a Mab-ZAP assay using an immunotoxin binding to thetherapeutic antibody wherein the toxin is released upon incorporationinto cells to inhibit cell growth (Bio Techniques 28: 162-165, January2000). As the immunotoxin, a recombinant complex protein of a diphtheriatoxin catalytic domain and protein G may be used.

In the present invention, “high internalization ability” refers to thesituation that the survival rate (which is a relative rate to the cellsurvival rate without addition of the antibody as 100%) of targetedantigen-expressing cells (e.g., CLDN6-expressing cells) with addition ofthe antibody and a saporin-labeled anti-mouse or rat IgG antibody ispreferably 70% or less, and more preferably 60% or less.

Since the compound conjugated in the antibody-drug conjugate of thepresent invention exerts an antitumor effect, it is preferred but notessential that the antibody itself should have an antitumor effect. Forthe purpose of specifically and selectively exerting the cytotoxicity ofthe antitumor compound against tumor cells, it is important and alsopreferred that the antibody should have the property of internalizing tomigrate into tumor cells. To exert antitumor effect, it is important andalso preferred that the antibody should have the property ofinternalizing and migrating into tumor cells, from the viewpoint thatthe drug specifically and selectively damages tumor cells. The antitumoractivity of the antibody refers to the cellular cytotoxic activity oranticellular effect against tumor cells. The antitumor activity may beconfirmed by using any known in vitro or in vivo evaluation system.

Examples of such an antibody may include, but not limited to, antibodiesto tumor-related antigens, including an anti-CLDN6 antibody, ananti-CLDN6/CLDN9 antibody, an anti-HER2 antibody, an anti-DLL3 (Deltalike protein 3) antibody, an anti-A33 antibody, an anti-CanAg antibody,an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, ananti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, ananti-CD70 antibody, an anti-CD98 antibody, an anti-TROP2 antibody, ananti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, ananti-FGFR2 antibody (e.g., WO 201315206), an anti-G250 antibody, ananti-MUC1 antibody (e.g., WO 2011012309), an anti-GPNMB antibody, ananti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-Cantibody, an anti-SLC44A4 antibody, an anti-mesothelin antibody, ananti-EGFR antibody, and an anti-DR5 antibody.

The antibody of the present invention is preferably an anti-CLDN6antibody, an anti-CLDN6/CLDN9 antibody, an anti-HER2 antibody, ananti-CD98 antibody, or an anti-TROP2 antibody, and more preferably ananti-CLDN6 antibody or an anti-HER2 antibody (e.g., trastuzumab, atrastuzumab variant).

The antibody of the present invention may be obtained using a methodusually carried out in the art, which involves immunizing animals withan antigenic polypeptide and collecting and purifying antibodiesproduced in vivo. The origin of the antigen is not limited to humans,and the animals may be immunized with an antigen derived from anon-human animal such as a mouse, a rat or the like. In this case, thecross-reactivity of antibodies binding to the obtained heterologousantigen with human antigens can be tested to screen for an antibodyapplicable to a human disease.

Alternatively, antibody-producing cells which produce antibodies againstthe antigen are fused with myeloma cells according to a method known inthe art (e.g., Nature (1975) 256, p. 495-497, Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)) to establish hybridomas, from whichmonoclonal antibodies can in turn be obtained (described later).

The antigen can be obtained by genetically engineering host cells toproduce a gene encoding the antigenic protein.

The chimeric antibody and humanized antibody of the present inventionmay be obtained in accordance with a known method (e.g., Proc. Natl.Acad. Sci. U.S.A., 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525,WO 90/07861).

The anti-HER2 antibody (e.g., U.S. Pat. No. 5,821,337), anti-TROP2antibody (e.g., WO 2003/074566), and anti-CD98 antibody (e.g., WO2015/146132) may be obtained by using a known approach.

Now, the anti-CLDN6 antibody used in the present invention will bedescribed. An embodiment described below is an example of representativeembodiments of the present invention, and the scope of the presentinvention is not interpreted as being narrower by the embodiment.

1. CLDN6 and CLDN9

CLDN6, a four-transmembrane protein belonging to the claudin family andconsisting of 220 amino acids, has the N terminus and C terminus in acell.

The amino acid sequence of and DNA sequence for human CLDN6 arepublished in public databases, and can be referred to, for example, fromaccession numbers of NP_067018 (SEQ ID NO: 1 (FIG. 11 )) and NM_021195(SEQ ID NO: 2 (FIG. 11 ) (both in NCBI).

In the amino acid sequence of human CLDN6 protein (hereinafter, referredto as “CLDN6 amino acid sequence”), the extracellular region is composedof an extracellular domain (EC1) consisting of amino acid residues 29 to81 of SEQ ID NO: 1 in Sequence Listing and an extracellular domain (EC2)consisting of amino acid residues 138 to 160 of SEQ ID NO: 1 in SequenceListing.

CLDN9, a four-transmembrane protein belonging to the claudin family andconsisting of 217 amino acids, has the N terminus and C terminus in acell. CLDN9 is highly homologous to CLDN6.

The amino acid sequence of and DNA sequence for human CLDN9 arepublished in public databases, and can be referred to, for example, fromaccession numbers of NP_066192 (SEQ ID NO: 3 (FIG. 12 )) and NM_020982(SEQ ID NO: 4 (FIG. 12 )) (both in NCBI).

2. Anti-CLDN6 Antibody

An example of the anti-CLDN6 antibody of the present invention is ananti-CLDN6 antibody that recognizes a higher order structure includingtwo extracellular regions, specifically, an amino acid sequence of the29- to 81-positions and amino acid sequence of the 138- to 160-positionsfrom the N terminus of CLDN6 as represented by SEQ ID NO: 1 in SequenceListing, and has internalization activity.

The anti-CLDN6 antibody of the present invention is an antibody capableof targeting tumor cells, and specifically has a property of recognizinga tumor cell, a property of binding to a tumor cell, a property of beingincorporated and internalizing in a tumor cell, and so on. Accordingly,the anti-CLDN6 antibody according to the present invention can be usedfor an antibody-drug conjugate by conjugating via a linker with acompound having antitumor activity.

The anti-CLDN6 antibody of the present invention may have antitumoractivity.

The anti-CLDN6 antibody may be obtained using a method usually carriedout in the art, which involves immunizing animals with an antigenicpolypeptide and collecting and purifying antibodies produced in vivo.CLDN6 is a four-transmembrane protein, and hence protein retaining thethree-dimensional structure may be used as an antigen, and examples ofsuch methods may include, but not limited to, cell immunization.

Alternatively, antibody-producing cells which produce antibodies againstthe antigen are fused with myeloma cells according to the method knownin the art to establish hybridomas, from which monoclonal antibodies canin turn be obtained.

Now, a method for obtaining an antibody against CLDN6 will bespecifically described.

1) Preparation of Antigen

CLDN6 may be directly purified for use from tumor tissue or tumor cellsof a human, or a cell membrane fraction of the cells may be prepared foruse as CLDN6. Alternatively, CLDN6 may be obtained by synthesizing CLDN6in vitro (e.g., Rapid Translation System (RTS) produced by RocheDiagnostics K.K.), or allowing host cells to produce CLDN6 through geneengineering.

To obtain the antigen through gene engineering, cDNA for CLDN6 isincorporated into a vector capable of expressing the cDNA, and CLDN6 issynthesized in a solution containing an enzyme, substrate, and energysubstance required for transcription and translation, or host cells ofanother prokaryote or eukaryote are transformed to allow the cells toexpress CLDN6. Alternatively, CLDN6-expressing cells obtained throughthe gene engineering or a cell line expressing CLDN6 may be used asCLDN6 protein.

The antigen may be obtained as a secretory protein by allowing anappropriate host-vector system to express a fusion protein including theextracellular region of the membrane protein CLDN6 and the constantregion of an antibody linked together.

The above-described transformant itself may be used as an antigen.

Further, a cell line that expresses CLDN6 may be used as the antigen.Examples of such cell lines may include cells of the human pancreaticcancer cell line NOR-P1; the human ovarian cancer cell linesNIH:OVCAR-3, OV-90, and OAW28; the human ovarian teratoma cell linePA-1; the human liver cancer cell line HuH-7; the human gastationalchoriocarcinoma cell line JEG-3; and human pluripotent embryoniccarcinoma cell line NTERA-2 clone DI, but are not limited thereto andany cell line that expresses CLDN6 is acceptable.

The CLDN9 protein to be used in the present invention may be preparedfor use in the same manner.

2) Production of Anti-CLDN6 Monoclonal Antibody

The anti-CLDN6 antibody used in the present invention is not limited toa particular antibody, and, for example, an antibody specified by any ofthe amino acid sequences listed in the present Sequence Listing can bepreferably used. The anti-CLDN6 antibody to be used in the presentinvention is desired to have the following properties.

(1) An antibody having the following properties (a) and (b).

(a) Recognizing or binding to the CLDN family.

The antibody of the present invention recognizes the CLDN family. Inother words, the antibody of the present invention binds to the CLDNfamily. The antibody of the present invention preferably binds to CLDN6,and more preferably specifically binds to CLDN6. Further, the antibodyof the present invention may recognize CLDN9 or bind to CLDN9.

In the present invention, “specific recognition”, that is, “specificbinding” refers to binding being not nonspecific adsorption. Examples ofdetermination criteria on whether binding is specific or not mayinclude, but not limited to, dissociation constants (hereinafter,referred to as “KD”). A preferred KD value of the antibody of thepresent invention to CLDN6 and/or CLDN9 is 1×10⁻⁵ M or less, 5×10⁻⁶ M orless, 2×10⁻⁶ M or less, or 1×10⁻⁶ M or less, and more preferably 5×10⁻⁷M or less, 2×10⁻⁷ M or less, or 1×10⁻⁷ M or less.

Binding between an antigen and an antibody in the present invention maybe measured or determined by an analysis method such as an ELISA method,an RIA method, and surface plasmon resonance (hereinafter, referred toas “SPR”). Binding between an antigen expressed on a cell surface and anantibody may be measured, for example, by a flow cytometry method.

(b) Having activity to internalize in CLDN6- and/or CLDN9-expressingcells through binding to CLDN6 and/or CLDN9.

(2) The antibody according to (1), wherein CLDN6 and/or CLDN9 are/ishuman CLDN6 and/or human CLDN9.

The method of the present invention for obtaining the antibody againstCLDN6 typically involves the following steps, but is not limited to thefollowing.

(Method Using Hybridoma)

(a) Purification of a biopolymer for use as the antigen or preparationof antigen-expressing cells, and administration of the biopolymer orantigen-expressing cells to an animal;

(b) collection of tissue (e.g., a lymph node) includingantibody-producing cells from the animal for which immunoreaction hasbeen induced;

(c) preparation of myeloma cells (e.g., mouse myeloma SP2/0-ag14 cells);

(d) cell fusion of antibody-producing cells and myeloma cells;

(e) selection of a hybridoma group producing the targeted antibody;

(f) division into single cell clones (cloning);

(g) an optional step of culture of the hybridoma for mass production ofan monoclonal antibody or rearing of an animal to which the hybridomawas transplanted; and

(h) examination of the physiological activity (internalization activity)and the binding specificity of the thus-produced monoclonal antibody, ortesting of properties as a labeling reagent.

Examples of methods to be used here for measuring antibody titers mayinclude, but not limited to, flow cytometry and a Cell-ELISA method.

Examples of the thus-obtained monoclonal anti-CLDN6 antibody mayinclude, but not limited to, the mouse anti-CLDN6 antibodies B1 and C7.In the present invention, the “B1” and the “C7” are occasionally calledas the “B1 antibody” and the “C7 antibody”, respectively.

The nucleotide sequence for and the amino acid sequence of the heavychain variable region of the B1 antibody are respectively represented bySEQ ID NO: 20 (FIG. 19 ) and SEQ ID NO: 21 (FIG. 19 ) in SequenceListing. The nucleotide sequence for and the amino acid sequence of thelight chain variable region of the B1 antibody are respectivelyrepresented by SEQ ID NO: 18 (FIG. 18 ) and SEQ ID NO: 19 (FIG. 18 ) inSequence Listing.

The amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3of the B1 antibody are represented by SEQ ID NO: 9 (FIG. 15 ), SEQ IDNO: 10 (FIG. 15 ), SEQ ID NO: 11 (FIG. 15 ), SEQ ID NO: 5 (FIG. 13 ),SEQ ID NO: 6 (FIG. 13 ), and SEQ ID NO: 7 (FIG. 13 ), respectively.

The nucleotide sequence for and the amino acid sequence of the heavychain variable region of the C7 antibody are respectively represented bySEQ ID NO: 24 (FIG. 21 ) and SEQ ID NO: 25 (FIG. 21 ) in SequenceListing. The nucleotide sequence for and the amino acid sequence of thelight chain variable region of the C7 antibody are respectivelyrepresented by SEQ ID NO: 22 (FIG. 20 ) and SEQ ID NO: 23 (FIG. 20 ) inSequence Listing.

The amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3of the C7 antibody are represented by SEQ ID NO: 15 (FIG. 17 ), SEQ IDNO: 16 (FIG. 17 ), SEQ ID NO: 17 (FIG. 17 ), SEQ ID NO: 12 (FIG. 16 ),SEQ ID NO: 13 (FIG. 16 ), and SEQ ID NO: 14 (FIG. 16 ), respectively.

Further, even if a monoclonal antibody was independently obtained bysteps (a) to (h) in “Production of anti-CLDN6 antibody” again, or amonoclonal antibody was separately obtained by using another method, anantibody having internalization activity equivalent to that of the B1antibody or C7 antibody can be obtained. An example of such antibodiesis an antibody that binds to an epitope for the B1 antibody or C7antibody. If a monoclonal antibody newly produced binds to a partialpeptide or partial three-dimensional structure to which the B1 antibodyor C7 antibody binds, it can be determined that the monoclonal antibodybinds to an epitope for the B1 antibody or C7 antibody. By confirmingthat the monoclonal antibody competes with the B1 antibody or C7antibody for binding to CLDN6 (i.e., the monoclonal antibody interfereswith binding between the B1 antibody or C7 antibody and CLDN6), it canbe determined, even when the specific sequence or structure of anepitope has not been determined, that the monoclonal antibody binds toan epitope for the anti-CLDN6 antibody. If epitope identity has beenconfirmed, the monoclonal antibody is strongly expected to haveantigen-binding ability, biological activity, and/or internalizationactivity equivalent to that of the B1 antibody or C7 antibody.

The antibody of the present invention includes, in addition to themonoclonal antibody against CLDN6, a gene recombinant antibody obtainedby artificial modification for the purpose of decreasing heterologousantigenicity to humans such as a chimeric antibody, a humanizedantibody, and a human antibody. These antibodies can be produced using aknown method.

(1) Chimeric Antibody

Examples of the chimeric antibody may include, but not limited to, anantibody in which antibody variable and constant regions are derivedfrom different species, for example, a chimeric antibody in which amouse- or rat-derived antibody variable region is connected to ahuman-derived antibody constant region (see Proc. Natl. Acad. Sci. USA,81, 6851-6855, (1984)).

A chimeric antibody derived from the mouse anti-human CLDN6 antibody B1antibody, as an example of the chimeric antibody of the presentinvention, is an antibody comprising a heavy chain comprising a heavychain variable region consisting of an amino acid sequence representedby SEQ ID NO: 21 (FIG. 19 ) and a light chain comprising a light chainvariable region represented by SEQ ID NO: 19 (FIG. 18 ), which maycomprising any human-derived constant region.

Specific examples of the chimeric antibody derived from the mouseanti-human CLDN6 antibody B1 antibody may include, but not limited to,the chimeric antibody chB1 antibody (hereinafter, also called as “chB1”)derived from the mouse anti-human CLDN6 antibody B1 antibody. Examplesof the chB1 antibody, in terms of the amino acid sequence, may include,but not limited to, an antibody comprising a heavy chain having an aminoacid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:32 (FIG. 24 ) in Sequence Listing and a light chain having an amino acidsequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 28(FIG. 22 ) in Sequence Listing.

In the heavy chain sequence represented by SEQ ID NO: 32 (FIG. 24 ) inSequence Listing, the amino acid sequence consisting of amino acidresidues 1 to 19 is the signal sequence, the amino acid sequenceconsisting of amino acid residues 20 to 141 is the heavy chain variableregion, and the amino acid sequence consisting of amino acid residues142 to 471 is the heavy chain constant region. In the light chainsequence represented by SEQ ID NO: 28 (FIG. 22 ) in Sequence Listing,the amino acid sequence consisting of amino acid residues 1 to 20 is thesignal sequence, the amino acid sequence consisting of amino acidresidues 21 to 127 is the light chain variable region, and the aminoacid sequence consisting of amino acid residues 128 to 234 is the lightchain constant region.

The amino acid sequences of the heavy chain and light chain variableregions of the chB1 antibody are respectively represented by SEQ ID NO:34 (FIG. 25 ) and SEQ ID NO: 30 (FIG. 23 ) in Sequence Listing.

The heavy chain amino acid sequence of the chB1 antibody is encoded by anucleotide sequence represented by SEQ ID NO: 33 (FIG. 24 ) in SequenceListing. A nucleotide sequence consisting of nucleotide residues 1 to 57of a nucleotide sequence represented by SEQ ID NO: 33 in SequenceListing is encoding the signal sequence of the chB1 antibody heavychain, a nucleotide sequence consisting of nucleotide residues 58 to 423of a nucleotide sequence represented by SEQ ID NO: 33 in SequenceListing is encoding the heavy chain variable region of the chB1antibody, and a nucleotide sequence consisting of nucleotide residues424 to 1413 of a nucleotide sequence represented by SEQ ID NO: 33 inSequence Listing is encoding the heavy chain constant region of the chB1antibody.

The nucleotide sequence for the heavy chain variable region of the chB1antibody is represented by SEQ ID NO: 35 (FIG. 25 ) in Sequence Listing.The light chain amino acid sequence of the chB1 antibody is encoded by anucleotide sequence represented by SEQ ID NO: 29 (FIG. 22 ) in SequenceListing. A nucleotide sequence consisting of nucleotide residues 26 to85 of a nucleotide sequence represented by SEQ ID NO: 29 in SequenceListing is encoding the signal sequence of the chB1 antibody lightchain, a nucleotide sequence consisting of nucleotide residues 86 to 406of a nucleotide sequence represented by SEQ ID NO: 29 in SequenceListing is encoding the light chain variable region of the chB1antibody, and a nucleotide sequence consisting of nucleotide residues407 to 727 of a nucleotide sequence represented by SEQ ID NO: 29 inSequence Listing is encoding the light chain constant region of the chB1antibody.

The nucleotide sequence for the light chain variable region of the chB1antibody is represented by SEQ ID NO: 31 (FIG. 23 ) in Sequence Listing.

(2) Humanized Antibody

Examples of the humanized antibody may include, but not limited to, anantibody obtained by incorporating only the complementarity determiningregions (CDRs) into a human-derived antibody (see Nature (1986) 321, p.522-525), an antibody obtained by grafting a part of the amino acidresidues of a framework as well as the CDR sequences to a human antibodyby a CDR-grafting method (WO 90/07861), and an antibody in which a partof the CDR amino acid sequences has been modified with the bindingability to an antigen maintained.

If the humanized antibody is derived from the B1 antibody or C1antibody, however, the humanized antibody may be any humanized antibody,without limited to a particular humanized antibody, that retains all thesix CDR sequences of the B1 antibody or C1 antibody and hasCLDN6-binding activity, and in addition the humanized antibody may beany humanized antibody, without limited to a particular humanizedantibody, such that its humanized antibody variant in which one toseveral (preferably, one or two, more preferably, one) CDR amino acidsequences have been modified also recognizes CLDN6 protein, or has theCLDN6 protein-binding activity of the original antibody.

Examples of the humanized anti-CLDN6 antibody of the present inventionor a functional fragment thereof may include, but not limited to, anantibody comprising a heavy chain having a variable region comprising:

CDRH1 consisting of an amino acid sequence represented by SEQ ID NO: 9(FIG. 15 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid sequence;

CDRH2 consisting of an amino acid sequence represented by SEQ ID NO: 10(FIG. 15 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid sequence; and

CDRH3 consisting of an amino acid sequence represented by SEQ ID NO: 11(FIG. 15 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid sequence; and

a light chain having a variable region comprising:

CDRL1 consisting of an amino acid sequence represented by SEQ ID NO: 5(FIG. 13 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid sequence;

CDRL2 consisting of an amino acid sequence represented by SEQ ID NO: 6(FIG. 13 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid sequence; and

CDRL3 consisting of an amino acid sequence represented by SEQ ID NO: 7(FIG. 13 ) in Sequence Listing, or an amino acid sequence obtained bysubstituting one to several (preferably, one or two) amino acids in theaforementioned amino acid, and

recognizing the CLDN6 protein of the present invention or retaining theCLDN6 protein-binding activity of the antibody,

or a functional fragment of the antibody.

Preferred examples of CDR amino acid substitution in the humanizedanti-CLDN6 antibody or functional fragment thereof may include, but notlimited to, substitution of one to several (preferably, one or two)amino acids in CDRL3 as described above, and an example thereof is CDRL3represented by SEQ ID NO: 8 (FIG. 14 ) in Sequence Listing, which isobtained by substituting amino acid residues 4 and 5 of SEQ ID NO: 7 inSequence Listing.

Examples of the heavy chain variable region of the humanized antibodycompising the above-described CDRHs may include, but not limited to, anamino acid sequence represented by SEQ ID NO: 54 (FIG. 35 ) in SequenceListing, an amino acid sequence represented by SEQ ID NO: 58 (FIG. 37 )in Sequence Listing, and an amino acid sequence represented by SEQ IDNO: 62 (FIG. 39 ) in Sequence Listing, and examples of the light chainvariable region of the humanized antibody compising the above-describedCDRLs may include, but not limited to, an amino acid sequencerepresented by SEQ ID NO: 38 (FIG. 27 ) in Sequence Listing, an aminoacid sequence represented by SEQ ID NO: 42 (FIG. 29 ) in SequenceListing, and an amino acid sequence represented by SEQ ID NO: 46 (FIG.31 ) in Sequence Listing.

Preferred examples of humanized antibodies including a combination ofthe above heavy chain variable region and light chain variable regionmay include, but not limited to:

a humanized antibody compising a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 54 (FIG. 35 ) inSequence Listing and a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 38 (FIG. 27 ) in SequenceListing;

a humanized antibody compising a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 58 (FIG. 37 ) inSequence Listing and a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 42 (FIG. 29 ) in SequenceListing;

a humanized antibody compising a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 54 (FIG. 35 ) inSequence Listing and a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 46 (FIG. 31 ) in SequenceListing;

a humanized antibody compising a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 58 (FIG. 37 ) inSequence Listing and a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 50 (FIG. 33 ) in SequenceListing; and

a humanized antibody compising a heavy chain variable region consistingof an amino acid sequence represented by SEQ ID NO: 62 (FIG. 39 ) inSequence Listing and a light chain variable region consisting of anamino acid sequence represented by SEQ ID NO: 46 (FIG. 31 ) in SequenceListing.

Examples of full-length sequences of humanized antibodies including acombination of the above heavy chain variable region and light chainvariable region may include, but not limited to:

a humanized antibody compising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 52(FIG. 34 ) in Sequence Listing and a light chain consisting of an aminoacid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:36 (FIG. 26 ) in Sequence Listing (H1L1);

a humanized antibody compising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 56(FIG. 36 ) in Sequence Listing and a light chain consisting of an aminoacid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:40 (FIG. 28 ) in Sequence Listing (H2L2);

a humanized antibody compising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 52(FIG. 34 ) in Sequence Listing and a light chain consisting of an aminoacid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:44 (FIG. 30 ) in Sequence Listing (H1L3);

a humanized antibody compising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 56(FIG. 36 ) in Sequence Listing and a light chain consisting of an aminoacid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:48 (FIG. 32 ) in Sequence Listing (H2L4); and

a humanized antibody compising a heavy chain consisting of an amino acidsequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 60(FIG. 38 ) in Sequence Listing and a light chain consisting of an aminoacid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:44 (FIG. 30 ) in Sequence Listing (H3L3).

In the heavy chain amino acid sequence represented by SEQ ID NO: 52(FIG. 34 ), 56 (FIG. 36 ), or 60 (FIG. 38 ) in Sequence Listing, anamino acid sequence consisting of amino acid residues 1 to 19 is thesignal sequence, an amino acid sequence consisting of amino acidresidues 20 to 141 is the heavy chain variable region, and an amino acidsequence consisting of amino acid residues 142 to 471 is the heavy chainconstant region.

In the light chain amino acid sequence represented by SEQ ID NO: 36(FIG. 26 ), 40 (FIG. 28 ), 44 (FIG. 30 ), or 48 (FIG. 32 ), an aminoacid sequence consisting of amino acid residues 1 to 20 is the signalsequence, an amino acid sequence consisting of amino acid residues 21 to127 is the light chain variable region, and an amino acid sequenceconsisting of amino acid residues 128 to 234 is the light chain constantregion.

The nucleotide sequence encoding the heavy chain amino acid sequence ofthe humanized antibody H1L1 and that encoding the light chain amino acidsequence of the humanized antibody H1L1 are a polynucleotide representedby SEQ ID NO: 53 (FIG. 34 ) and a polynucleotide represented by SEQ IDNO: 37 (FIG. 26 ), respectively;

the nucleotide sequence encoding the heavy chain amino acid sequence ofthe humanized antibody H2L2 and that encoding the light chain amino acidsequence of the humanized antibody H2L2 are a polynucleotide representedby SEQ ID NO: 57 (FIG. 36 ) and a polynucleotide represented by SEQ IDNO: 41 (FIG. 28 ), respectively;

the nucleotide sequence encoding the heavy chain amino acid sequence ofthe humanized antibody H1L3 and that encoding the light chain amino acidsequence of the humanized antibody H1L3 are a polynucleotide representedby SEQ ID NO: 53 (FIG. 34 ) and a polynucleotide represented by SEQ IDNO: 45 (FIG. 30 ), respectively;

the nucleotide sequences encoding the heavy chain amino acid sequence ofthe humanized antibody H2L4 and that encoding the light chain amino acidsequence of the humanized antibody H2L4 are a polynucleotide representedby SEQ ID NO: 57 (FIG. 36 ) and a polynucleotide represented by SEQ IDNO: 49 (FIG. 32 ), respectively; and

the nucleotide sequence encoding the heavy chain amino acid sequence ofthe humanized antibody H3L3 and that encoding the light chain amino acidsequence of the humanized antibody H3L3 are a polynucleotide representedby SEQ ID NO: 61 (FIG. 38 ) and a polynucleotide represented by SEQ IDNO: 45 (FIG. 30 ), respectively.

The nucleotide sequence encoding the amino acid sequence of the heavychain variable region of the humanized antibody H1L1 and that encodingthe light chain variable region of the humanized antibody H1L are apolynucleotide represented by SEQ ID NO: 55 (FIG. 35 ) and apolynucleotide represented by SEQ ID NO: 39 (FIG. 27 ), respectively;

the nucleotide sequence encoding the amino acid sequence of the heavychain variable region of the humanized antibody H2L2 and that encodingthe light chain variable region of the humanized antibody H2L2 are apolynucleotide represented by SEQ ID NO: 59 (FIG. 37 ) and apolynucleotide represented by SEQ ID NO: 43 (FIG. 29 ), respectively;

the nucleotide sequence encoding the amino acid sequence of the heavychain variable region of the humanized antibody H1L3 and that encodingthe light chain variable region of the humanized antibody H1L3 are apolynucleotide represented by SEQ ID NO: 55 (FIG. 35 ) and apolynucleotide represented by SEQ ID NO: 47 (FIG. 31 ), respectively;

the nucleotide sequence encoding the amino acid sequence of the heavychain variable region of the humanized antibody H2L4 and that encodingthe light chain variable region of the humanized antibody H2L4 are apolynucleotide represented by SEQ ID NO: 59 (FIG. 37 ) and apolynucleotide represented by SEQ ID NO: 51 (FIG. 33 ), respectively;and

the nucleotide sequence encoding the amino acid sequence of the heavychain variable region of the humanized antibody H3L3 and that encodingthe light chain variable region of the humanized antibody H3L3 are apolynucleotide represented by SEQ ID NO: 63 (FIG. 39 ) and apolynucleotide represented by SEQ ID NO: 47 (FIG. 31 ), respectively.

In the nucleotide sequence represented by SEQ ID NO: 53 (FIG. 34 ), 57(FIG. 36 ), or 61 (FIG. 38 ) in Sequence Listing, a nucleotide sequenceconsisting of nucleotide resides 1 to 57 is encoding the signal sequenceof the humanized antibody heavy chain, a nucleotide sequence consistingof nucleotide resides 58 to 423 is encoding the amino acid sequence ofthe variable region of the humanized antibody heavy chain, and anucleotide sequence consisting of nucleotide resides 424 to 1413 isencoding the constant region of the antibody heavy chain.

In the nucleotide sequence represented by SEQ ID NO: 37 (FIG. 26 ), 41(FIG. 28 ), 45 (FIG. 30 ), or 49 (FIG. 32 ) in Sequence Listing, anucleotide sequence consisting of nucleotide resides 1 to 60 is encodingthe signal sequence of the humanized antibody light chain, a nucleotidesequence consisting of nucleotide residues 61 to 381 is encoding theamino acid sequence of the variable region of the humanized antibodylight chain, and a nucleotide sequence consisting of nucleotide residues382 to 702 is encoding the constant region of the antibody light chain.

As long as having binding activity to CLDN6, any antibody that has anidentity or homology of 80% or higher, preferably of 90% or higher, morepreferably of 95% or higher, even more preferably of 97% or higher, themost preferably of 99% or higher, to the amino acid sequence of any ofthe antibodies including the above combinations of a heavy chainvariable region and a light chain variable region and the antibodiesincluding the above combinations of a heavy chain and a light chain isalso included in the antibody of the present invention.

As long as having binding activity to CLDN6, any antibody that includesCDRs consisting of the amino acid sequences of the CDRs of any of theantibodies including the above combinations of a heavy chain variableregion and a light chain variable region and the antibodies includingthe above combinations of a heavy chain and a light chain, wherein theamino acid sequence of the antibody excluding the amino acid sequencesof the CDRs has an amino acid identity or homology of 80% or higher,preferably of 90% or higher, more preferably of 95% or higher, even morepreferably of 97% or higher, the most preferably of 99% or higher, isalso included in the antibody of the present invention.

Further, an antibody having biological activity equivalent to each ofthe above antibodies may be selected through combining amino acidsequences obtained by substituting, deleting, or adding one or severalamino acid residues in the amino acid sequence of the heavy chain orlight chain. The substitution of an amino acid herein is preferablyconservative amino acid substitution (WO 2013154206).

The conservative amino acid substitution is substitution that occurs inan amino acid group with related amino acid side chains. Such amino acidsubstitution is preferably carried out to such a degree that theproperties of the substance having the original amino acid sequence arenot decreased.

Homology between two amino acid sequences may be determined by usingdefault parameters of Blast algorithm version 2.2.2 (Altschul, StephenF., Thomas L. Madden, Alejandro A. Schaaffer, Jinghui Zhang, ZhengZhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25: 3389-3402) Blast algorithm may be used byaccessing www.ncbi.nlm.nih.gov/blast on the Internet.

(3) Human Antibody

Further examples of the antibody of the present invention may include,but not limited to, human antibodies capable of binding to CLDN6 and/orCLDN9. The human anti-CLDN6 and/or CLDN9 antibody refers to a humanantibody having only an antibody gene sequence derived from a humanchromosome. The human anti-CLDN6 antibody may be obtained by using amethod with a human antibody-producing mouse having a human chromosomefragment including the genes of a heavy chain and light chain of a humanantibody (see Nature Genetics (1997) 16, p. 133-143; Nucl. Acids Res.(1998) 26, p. 3447-3448; Animal Cell Technology: Basic and AppliedAspects vol. 10, p. 69-73, Kluwer Academic Publishers, 1999; Proc. Natl.Acad. Sci. USA (2000) 97, p. 722-727, etc.).

Specifically, such a human antibody-producing mouse may be created byproducing a knockout animal or transgenic animal as a gene recombinantanimal with the gene loci for the heavy chain and light chain ofendogenous immunoglobulin destroyed, instead, with the gene loci for theheavy chain and light chain of human immunoglobulin introduced therein,for example, via a yeast artificial chromosome (YAC) vector, andinterbreeding of such animals.

Alternatively, such an antibody may be obtained as follows: a eukaryoticcell is transformed with cDNA encoding the heavy chain and light chainof a human antibody, preferably with a vector including the cDNA,through a gene recombinant technique, and the transformed cell producinga gene recombinant human monoclonal antibody is cultured, and theantibody is obtained from the culture supernatant.

For the host, for example, a eukaryotic cell, preferably a mammaliancell such as a CHO cell, a lymphocyte, and a myeloma cell may be used.

In addition, a method of obtaining a phage display-derived humanantibody sorted out of a human antibody library (see InvestigativeOphthalmology & Visual Science (2002) 43 (7), p. 2301-2308; Briefings inFunctional Genomics and Proteomics (2002), 1 (2), p. 189-203;Ophthalmology (2002) 109 (3), p. 427-431, etc.) is known.

For example, a phage display method (Nature Biotechnology (2005), 23,(9), p. 1105-1116) may be used, in which the variable region of a humanantibody is expressed as a single chain antibody (scFv) on phagesurfaces, and phages that bind to the antigen are selected.

Analysis of phage genes selected because of binding to the antigen candetermine the DNA sequence encoding the variable region of the humanantibody that binds to the antigen.

Once the DNA sequence of scFv that binds to the antigen has beenclarified, the human antibody can be obtained by producing an expressionvector including the sequence and introducing the expression vector intoan appropriate host for expression (WO92/01047, WO92/20791, WO93/06213,WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol(1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116)

Chimeric antibodies, humanized antibodies, human antibodies, and so onobtained by using the above method may be evaluated for binding activityto an antigen, for example, by using a known method to screen for apreferred antibody.

Another example of indicators in comparing characteristics amongantibodies is stability of antibodies. Differential scanning calorimetry(DSC) is an apparatus capable of quickly and accurately measuringthermal denaturation midpoints (Tm), a good indicator for relativestructural stability of protein. Difference in thermal stability can becompared through comparison of Tm values measured with DSC. Storagestability of antibodies is known to be correlated with thermal stabilityof antibodies to some degree (Pharmaceutical Development and Technology(2007) 12, p. 265-273), and hence thermal stability may be used as anindicator to screen for a preferred antibody. Examples of otherindicators for screening for an antibody may include, but not limitedto, a high yield in appropriate host cells and a low agglutinatingproperty in aqueous solution. It is needed to screen for the mostsuitable antibody for administration to humans through comprehensivedetermination based on the above-described indicators, for example,because an antibody with the highest yield does not necessarily exhibitthe highest thermal stability.

The antibody of the present invention includes “antibodies that bind toa site to which the anti-CLDN6 antibody provided by the presentinvention binds”. That is, the present invention includes antibodiesthat bind to a site on CLDN6 protein that B1 or C7 of the presentinvention recognizes.

The antibody of the present invention includes modified variants of theantibody. The modified variant refers to a variant obtained bysubjecting the antibody of the present invention to chemical orbiological modification. Examples of the chemically modified variant mayinclude, but not limited to, variants including a linkage of a chemicalmoiety to an amino acid skeleton, and variants with chemicalmodification of an N-linked or O-linked carbohydrate chain. Examples ofthe biologically modified variant may include, but not limited to,variants obtained by post-translational modification (e.g., N-linked orO-linked glycosylation, N- or C-terminal processing, deamidation,isomerization of aspartic acid, oxidation of methionine), and variantsin which a methionine residue has been added to the N terminus by beingexpressed in a prokaryotic host cell. Further, an antibody labeled so asto enable the detection or isolation of the antibody of the presentinvention or an antigen, for example, an enzyme-labeled antibody, afluorescence-labeled antibody, and an affinity-labeled antibody are alsoincluded in the meaning of the modified variant. Such a modified variantof the antibody of the present invention is useful for improving thestability and blood retention of the antibody, reducing the antigenicitythereof, detecting or isolating an antibody or an antigen, and so on.

Further, by regulating the modification of a glycan which is linked tothe antibody of the present invention (glycosylation, defucosylation,etc.), the antibody-dependent cellular cytotoxic activity can beenhanced. As the technique for regulating the modification of a glycanof antibodies, WO 1999/54342, WO 2000/61739, WO 2002/31140, etc., areknown. However, the technique is not limited thereto. In the antibody ofthe present invention, antibodies in which the modification of a glycanis regulated are also included.

Such modification may be applied at any position or a desired positionin an antibody or a functional fragment of the antibody, and the sametype or two or more different types of modification may be applied atone or two or more positions.

In the present invention, the meaning of a “modified variant of anantibody fragment” also includes a “fragment of a modified variant of anantibody”.

If an antibody gene is temporarily isolated and then introduced into anappropriate host to produce an antibody, an appropriate combination of ahost and an expression vector can be used. Specific examples of theantibody gene may include, but not limited to, combination of a geneencoding the heavy chain sequence or the like of an antibody describedherein and a gene encoding the light chain sequence or the like of anantibody described herein. To transform host cells, a heavy chainsequence gene or the like and a light chain sequence gene or the likemay be inserted into the same expression vector, or inserted intoseparate expression vectors.

If eukaryotic cells are used as a host, animal cells, plant cells, andeukaryotic microorganisms may be used. Particularly, examples of animalcells may include, but not limited to, mammalian cells, such as COScells (Cell (1981) 23, p. 175-182, ATCC CRL-1650), as monkey cells, themouse fibroblast NIH3T3 (ATCC No. CRL-1658), a dihydrofolatereductase-deficient strain (Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p.4126-4220) of Chinese hamster ovary cells (CHO cells, ATCC CCL-61), andFreeStyle 293F cells (Invitrogen).

If prokaryotic cells are used, for example, Escherichia coli or Bacillussubtilis may be used.

A targeted antibody gene is introduced into these cells bytransformation, and the transformed cells are cultured in vitro toafford an antibody. Sequence difference among antibodies may result indifferent yields in the culture, and hence antibodies that allow easyproduction of a medicine may be selected out of antibodies havingequivalent binding activity by using yields as an indicator.Accordingly, the antibody of the present invention includes antibodiesobtained by using a method for producing the antibody, the methodincluding the steps of: culturing the transformed host cell; andcollecting a targeted antibody or a functional fragment of the antibodyfrom a culture obtained in the step of culturing.

The antibody gene is preferably a polynucleotide including apolynucleotide described in any one of (a) to (e):

(a) a combination of a polynucleotide encoding the heavy chain aminoacid sequence and a polynucleotide encoding the light chain amino acidsequence of an antibody of any one of the B1 or C7 antibody, the chB1antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3;(b) a combination of a polynucleotide encoding a heavy chain amino acidsequence including the sequences of CDRH1 to CDRH3 and a polynucleotideencoding a light chain amino acid sequence including the sequences ofCDRL1 to CDRL3 of an antibody of any one of the B1 or C7 antibody, thechB1 antibody, and the humanized antibodies H1L1, H2L2, H1L3, H2L4, andH3L3;(c) a combination of a polynucleotide encoding a heavy chain amino acidsequence comprising the amino acid sequence of the heavy chain variableregion and a polynucleotide encoding a light chain amino acid sequencecomprising the amino acid sequence of the light chain variable region ofan antibody of any one of the B1 or C7 antibody, the chB1 antibody, andthe humanized antibodies H1L1, H2L2, H1L3, H2L4, and H3L3;(d) a polynucleotide that is hybridizable with nucleotides consisting ofa polynucleotide complementary to the polynucleotide according to anyone of (a) to (c) under stringent conditions and is encoding the aminoacid sequence of an antibody capable of binding to CDLN6; and(e) a polynucleotide encoding the amino acid sequence of a polypeptideobtained by substituting, deleting, adding, or inserting 1 to 50, 1 to45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, oneto eight, one to six, one to five, one to four, one to three, one ortwo, or one amino acid(s) in the polynucleotide according to any one of(a) to (c), and is encoding the amino acid sequence of an antibodycapable of binding to CDLN6.

The present invention includes a nucleotide encoding the antibody of thepresent invention or a functional fragment of the antibody, or amodified variant of the antibody or functional fragment; a recombinantvector including the gene inserted therein; and a cell including thegene or the vector introduced therein.

The present invention includes a method for producing an antibody or afunctional fragment of the antibody, or a modified variant of theantibody or functional fragment, the method including the steps of:culturing the cell; and collecting from the culture an antibody or afunctional fragment of the antibody, or a modified variant of theantibody or functional fragment.

It is known that a lysine residue at the carboxyl terminus of the heavychain of an antibody produced in a cultured mammalian cell is deleted(Journal of Chromatography A, 705: 129-134 (1995)), and it is also knownthat two amino acid residues, glycine and lysine, at the carboxylterminus of the heavy chain of an antibody produced in a culturedmammalian cell are deleted and a proline residue newly located at thecarboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83(2007)). However, such deletion and modification of the heavy chainsequence do not affect the antigen-binding ability and the effectorfunction (the activation of complement, antibody-dependent cellularcytotoxicity, etc.) of the antibody. Therefore, in the antibodyaccording to the present invention, antibodies subjected to suchmodification and functional fragments of the antibody are also included,and deletion variants in which one or two amino acids have been deletedat the carboxyl terminus of the heavy chain, variants obtained byamidation of deletion variants (for example, a heavy chain in which thecarboxyl terminal proline residue has been amidated), and the like arealso included. The type of deletion variants having a deletion at thecarboxyl terminus of the heavy chain of the antibody according to thepresent invention is not limited to the above variants as long as theantigen-binding ability and the effector function are conserved. The twoheavy chains constituting the antibody according to the presentinvention may be of one type selected from the group consisting of afull-length heavy chain and the above-described deletion variant, or maybe of two types in combination selected therefrom. The ratio of theamount of each deletion variant can be affected by the type of culturedmammalian cells which produce the antibody according to the presentinvention and the culture conditions; however, an antibody in which oneamino acid residue at the carboxyl terminus has been deleted in both ofthe two heavy chains in the antibody according to the present inventioncan be preferably exemplified as a main component of molecules of theantibody.

Examples of isotypes of the anti-CLDN6 antibody of the present inventionmay include, but not limited to, IgG (IgG1, IgG2, IgG3, IgG4), andpreferred examples thereof include IgG1, IgG2, and IgG4.

If IgG1 is used as the isotype of the antibody of the present invention,the effector function may be adjusted by substituting some amino acidresidues in the constant region. Examples of variants of IgG1 with theeffector function lowered or attenuated may include, but not limited to,IgG1 LALA (IgG1-L234A, L235A) and IgG1 LAGA (IgG1-L235A, G237A), and apreferred variant of IgG1 is IgG1 LALA. The L234A, L235A indicatessubstitution of leucine with alanine at the 234- and 235-positionsspecified by EU-index numbering (Proc. Natl. Acad. Sci. U.S.A., Vol. 63,No. 1 (May 15, 1969), pp. 78-85), and the G237A indicates substitutionof glycine with alanine at the 237-position specified by EU-indexnumbering.

Typical examples of bioactivity of antibodies may include, but notlimited to, antigen-binding activity, activity to internalize in cellsexpressing an antigen by binding to the antigen, activity to neutralizeantigen activity, activity to enhance antigen activity,antibody-dependent cellular cytotoxicity (ADCC), complement-dependentcytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP),and the function of the antibody according to the present invention isbinding activity to CLDN6, and preferably activity to internalize inCLDN6-expression cells by binding to CLDN6. In addition to cellularinternalization activity, the antibody of the present invention may haveactivities of ADCC, CDC, and/or ADCP in combination.

The antibody obtained may be purified to a homogeneous state. Forseparation/purification of the antibody, separation/purification methodscommonly used for protein can be used. For example, the antibody may beseparated/purified by appropriately selecting and combining columnchromatography, filter filtration, ultrafiltration, salting-out,dialysis, preparative polyacrylamide gel electrophoresis, isoelectricfocusing, and so on (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual, Daniel R. Marshak et al.eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988)), but separation/purification methods are not limitedthereto.

Examples of chromatography may include, but not limited to, affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,gel filtration chromatography, reversed-phase chromatography, andadsorption chromatography.

These chromatographies may be carried out using liquid chromatographysuch as HPLC and FPLC.

Examples of columns for affinity chromatography may include, but notlimited to, a Protein A column and a Protein G column.

Alternatively, the antibody may be purified by utilizing bindingactivity to an antigen with a carrier to which the antigen has beenimmobilized.

It is desirable that the anti-HER2 antibody of the present invention be,for example, that having any of the following properties, but theanti-HER2 antibody is not limited thereto.

(1) An anti-HER2 antibody having the following properties:

(a) specifically binding to HER2; and

(b) internalizing into HER2-expressing cells by binding to HER2.

(2) The antibody according to (1), binding to the extracellular domainof HER2.

(3) The antibody according to (1) or (2), being a monoclonal antibody.

(4) The antibody according to any one of (1) to (3), having activitiesor activity of antibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC).

(5) The antibody according to any one of (1) to (4), being a mousemonoclonal antibody, a chimeric monoclonal antibody, or a humanizedmonoclonal antibody.

(6) The antibody according to any one of (1) to (3), wherein the heavychain constant region is a heavy chain constant region of human IgG1,and comprises a mutation that causes lowering of activities or activityof ADCC and/or CDC.

(7) The antibody according to (6), wherein the heavy chain constantregion is a heavy chain constant region of human IgG1, and leucine atthe 234- and 235-positions specified by EU Index numbering issubstituted with alanine.

(8) The antibody according to any one of (1) to (4), being a humanizedmonoclonal antibody comprising a heavy chain consisting of an amino acidsequence represented by SEQ ID NO: 65 and a light chain consisting of anamino acid sequence represented by SEQ ID NO: 64.(9) The antibody according to any one of (1) to (3), (6), and (7), beinga humanized monoclonal antibody comprising a heavy chain consisting ofan amino acid sequence consisting of amino acid residues 20 to 469 ofSEQ ID NO: 75 and a light chain consisting of an amino acid sequenceconsisting of amino acid residues 21 to 234 of SEQ ID NO: 73.(10) The antibody according to any one of (1) to (9), wherein one or twoamino acids are deleted at the carboxyl terminus of the heavy chain.(11) The antibody according to any one of (1) to (3), (8), and (10),comprising a heavy chain consisting of an amino acid sequence consistingof amino acid residues 1 to 449 of SEQ ID NO: 65 and a light chainconsisting of an amino acid sequence consisting of amino acid residues 1to 214 of SEQ ID NO: 64.(12) The antibody according to any one of (1) to (3), (6), (7), (9), and(10), comrising a heavy chain consisting of an amino acid sequenceconsisting of amino acid residues 20 to 468 of SEQ ID NO: 75 and a lightchain consisting of an amino acid sequence consisting of amino acidresidues 21 to 234 of SEQ ID NO: 73.(13) An antibody obtained by using a method for producing the antibodyaccording to any one of (1) to (12), the method including the steps of:culturing a host cell transformed with an expression vector containing apolynucleotide encoding the antibody; and collecting the targetedantibody from a culture obtained from the step of culturing.<Glycan Remodeling>

Recently has been reported a method for remodeling heterogeneousglycoprotein of an antibody by enzymatic reaction or the like tohomogeneously introduce a glycan having a functional group (ACS ChemicalBiology 2012, 7, 110, ACS Medicinal Chemistry Letters 2016, 7, 1005). Anattempt with use of this glycan remodeling technique has been made tosite-specifically introduce a drug to synthesize a homogeneous ADC(Bioconjugate Chemistry 2015, 26, 2233, Angew. Chem. Int. Ed. 2016, 55,2361-2367, US 2016361436).

In the glycan remodeling of the present invention, using hydrolase,heterogeneous glycans added to a protein (e.g., an antibody) are cleavedoff to leave only GlcNAc at each terminus thereby producing a homogenousprotein moiety with GlcNAc (hereinafter, referred to as an “acceptor”).Subsequently, an arbitrary glycan separately prepared (hereinafter,referred to as a “donor”) is provided, and the acceptor and the donorare linked together by using transglycosidase. Thereby, a homogeneousglycoprotein with arbitrary glycan structure can be synthesized.

In the present invention, a “glycan” refers to a structural unit of twoor more monosaccharides bonded together via glycosidic bonds. Specificmonosaccharides and glycans are occasionally abbreviated, for example,as “GlcNAc-”, “MSG-”, and so on. When any of these abbreviations is usedin a structural formula, the abbreviation is shown with an intentionthat an oxygen atom or nitrogen atom involved in a glycosidic bond atthe reducing terminal to another structural unit is not included in theabbreviation indicating the glycan, unless specifically defined.

In the present invention, a monosaccharide as a basic unit of a glycanis indicated for convenience so that in the ring structure, the positionof a carbon atom bonding to an oxygen atom constituting the ring anddirectly bonding to a hydroxy group (or an oxygen atom involved in aglycosidic bond) is defined as the 1-position (the 2-position only forsialic acids), unless otherwise specified. The names of compounds inExamples are each provided in view of the chemical structure as a whole,and that rule is not necessarily applied.

When a glycan is indicated as a sign (e.g., GLY, SG, MSG, GlcNAc) in thepresent invention, the sign is intended, unless otherwise defined, toinclude carbon atoms ranging to the reducing terminal and not to includeN or O involved in an N- or O-glycosidic bond.

In the present invention, unless specifically stated, a partialstructure when a glycan is linking to a side chain of an amino acid isindicated in such a manner that the side chain portion is indicated inparentheses, for example, “(SG-)Asn”.

The antibody-drug conjugate of the present invention is represented bythe following formula:

wherein antibody Ab or a functional fragment of the antibody may bondfrom a side chain of an amino acid residue thereof (e.g., cysteine,lysine) directly to L, or bond via a glycan or remodeled glycan of Ab toL, and preferably bonds via a glycan or remodeled glycan of Ab to L, andmore preferably bonds via a remodeled glycan of Ab to L.

Glycans in Ab of the present invention are N-linked glycans or O-linkedglycans, and preferably N-linked glycans.

N-linked glycans and O-linked glycans bond to an amino acid side chainof an antibody via an N-glycosidic bond and an O-glycosidic bond,respectively.

Ab of the present invention is IgG, and preferably IgG1, IgG2, or IgG4.

IgG has a well conserved N-linked glycan on an asparagine residue at the297-position of the Fc region of the heavy chain (hereinafter, referredto as “Asn297 or N297”), and the N-linked glycan is known to contributeto the activity and kinetics of the antibody molecule.

(Biotechnol. Prog., 2012, 28, 608-622, Sanglier-Cianferani, S., Anal.Chem., 2013, 85, 715-736)

The amino acid sequence in the constant region of IgG is well conserved,and each amino acid is specified by Eu index numbering in Edelman et al.(Proc. Natl. Acad. Sci. U.S.A., Vol. 63, No. 1 (May 15, 1969), pp.78-85). For example, Asn297, to which an N-linked glycan is added in theFc region, corresponds to the 297-position in Eu index numbering, andeach amino acid is uniquely specified by Eu index numbering, even if theactual position of the amino acid has varied through fragmentation ofthe molecule or deletion of a region.

In the antibody-drug conjugate of the present invention, the antibody orfunctional fragment of the antibody more preferably bonds to L via aglycan bonding to a side chain of Asn297 thereof (hereinafter, referredto as “N297 glycan”), and the antibody or functional fragment of theantibody even more preferably bonds via the N297 glycan to L, whereinthe N297 glycan is a remodeled glycan.

The following formula illustrates the situation that the antibody-drugconjugate of the present invention or a functional fragment of theantibody bonds via the N297 glycan to L.

An antibody having the remodeled glycan is referred to as aglycan-remodeled antibody.

SGP, an abbreviation for sialyl glycopeptide, is a representativeN-linked complex glycan. SGP can be separated/purified from the yolk ofa hen egg, for example, by using a method described in WO 2011/0278681.Purified products of SGP are commercially available (Tokyo ChemicalIndustry Co., Ltd., FUSHIMI Pharmaceutical Co., Ltd.), and may bepurchased. For example, disialooctasaccharide (Tokyo Chemical IndustryCo., Ltd.), a glycan formed by deleting one GlcNAc at the reducingterminal in the glycan moiety of SG (hereinafter, referred to as “SG(10)”, is commercially available.

In the present invention, a glycan structure formed by deleting a sialicacid at a non-reducing terminal only in either one of the branchedchains of β-Man in SG (10) refers to MSG (9), and a structure having asialic acid only in the 1-3 branched chain is called as MSG1, and astructure having a sialic acid only in the 1-6 branched chain is calledas MSG2.

The remodeled glycan of the present invention is N297-(Fuc)MSG1,N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, orN297-(Fuc)SG, and is preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, orN297-(Fuc)SG, and is more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

N297-(Fuc)MSG1 is represented by the following structural formula orsequence formula:

In the formulas, each wavy line represents bonding to Asn297 of theantibody,

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end represents amide-bonding to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in the 1-3branched chain of 1-Man in the N297 glycan,

each asterisk represents bonding to linker L, in particular, a nitrogenatom at the 1- or 3-position of the 1,2,3-triazole ring of Lb in linkerL, and

n⁵ is an integer of 2 to 10, and preferably an integer of 2 to 5.

N297-(Fuc)MSG2 is represented by the following structural formula orsequence formula:

In the formulas, each wavy line represents bonding to Asn297 of theantibody,

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end represents amide-bonding to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in the 1-6branched chain of β-Man in the N297 glycan,

each asterisk represents bonding to linker L, in particular, a nitrogenatom at the 1- or 3-position of the 1,2,3-triazole ring of Lb in linkerL, and

n⁵ is an integer of 2 to 10, and preferably an integer of 2 to 5.

N297-(Fuc)SG is represented by the following structural formula orsequence formula:

In the formulas, each wavy line represents bonding to Asn297 of theantibody,

L(PEG) represents —(CH₂CH₂—O)n⁵-CH₂CH₂—NH—, wherein the amino group atthe right end represents amide-bonding to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in each of the1-3 and 1-6 branched chains of β-Man in the N297 glycan,

each asterisk represents bonding to linker L, in particular, a nitrogenatom at the 1- or 3-position of the 1,2,3-triazole ring of Lb in linkerL, and

n⁵ is an integer of 2 to 10, and preferably an integer of 2 to 5.

If N297 glycan of the antibody in the antibody-drug conjugate of thepresent invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture ofthem, the antibody-drug conjugate is a molecule to which two moleculesof linker L and two molecules of drug D have been conjugated (m²=1)since the antibody is a dimer (see FIG. 1 ).

For example, Example 74: ADC8 is in the case that N297 glycan isN297-(Fuc)MSG1, and Example 67: ADC1 is in the case that N297 glycan isa mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2.

If N297 glycan of the antibody in the antibody-drug conjugate of thepresent invention is N297-(Fuc)SG, the antibody-drug conjugate is amolecule to which four molecules of linker L and four molecules of drugD have been conjugated (m²=2) since the antibody is a dimer. Forexample, Example 72: ADC6 is in the case that N297 glycan isN297-(Fuc)SG.

N297 glycan is preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, orN297-(Fuc)SG, and more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

If N297 glycan of the antibody in the antibody-drug conjugate of thepresent invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, ahomogeneous ADC can be obtained.

The present invention provides a method for producing a remodeledantibody or a functional fragment of the antibody, the method includingthe following steps of:

i) culturing the host cell (e.g., an animal cell (such as a CHO cell))according to any one of [46] to [48] and collecting a targeted antibodyfrom a culture obtained;

ii) treating the antibody obtained in step i) with hydrolase to producean antibody with N297 glycan being (Fucα1,6)GlcNAc((Fucα1,6)GlcNAc-antibody) (FIG. 3A);

preferably further purifying the (Fucα1,6)GlcNAc-antibody through a stepincluding purification of the reaction solution with a hydroxyapatitecolumn; and

any one of iii)-1 and iii)-2 (FIG. 3B):

iii)-1 reacting the (Fucα1,6)GlcNAc-antibody with a glycan donnermolecule in the presence of transglycosidase to synthesize aglycan-remodeled antibody with an azide group introduced to a sialicacid, the glycan donner molecule obtained by introducing a PEG linkerhaving an azide group (N₃-L(PEG)) to the carbonyl group of carboxylicacid at the 2-position of a sialic acid in MSG (9) or SG (10) andoxazolinating the reducing terminal; andiii)-2 reacting the (Fucα1,6)GlcNAc-antibody with a glycan donnermolecule in the presence of transglycosidase to synthesize aglycan-remodeled antibody with an azide group introduced to a sialicacid, the glycan donner molecule obtained by introducing a PEG linkerhaving an azide group (N₃-L(PEG)) to the carbonyl group of carboxylicacid at the 2-position of a sialic acid in (MSG-)Asn or (SG-)Asn with anα-amino group optionally protected or modified and to the carbonyl groupof carboxylic acid in the Asn, utilizing hydrolase, and thenoxazolinating the reducing terminal.

The present invention includes glycan-remodeled antibodies andfunctional fragments of the antibodies, and modified variants of theantibodies and functional fragments obtained by using the productionmethod.

The production intermediate of the present antibody-drug conjugate hasan alkyne structure reactive with an azide group, such as DBCO(dibenzocyclooctyne). Therefore, the antibody-drug conjugate of thepresent invention can be produced by reacting the productionintermediate with an MSG1-type, MSG2-type, or SG-type glycan-remodeledantibody or a functional fragment of the antibody, where the antibody,in which a PEG linker having an azide group has been introduced to asialic acid of a glycan, is obtained through steps i) to iii).

With regard to N297 glycan in the present invention, fucosylated GlcNAc((Fucα1,6)GlcNAc) at the reducing terminal is preferably derived from anantibody produced in an animal cell, and a portion of the glycan locatedto the non-reducing terminal side of (Fucα1,6)GlcNAc preferably has beenremodeled into the above-described glycan structure as MSG (MSG1, MSG2)or SG. In each case, carboxylic acid bonding to the 2-position of asialic acid at the non-reducing terminal is used for bonding to L(PEG).

Such a glycan-remodeled antibody having MSG- (MSG1-, MSG2-) or SG-typeN297 glycan may be produced by using a method as illustrated in FIGS. 3Aand 3B, for example, in accordance with a method described in WO2013/120066. If an antibody is produced as a gene-recombinant protein byusing an animal cell as a host in accordance with a known method (stepi), the N297 glycan has, as a base structure, a fucosylated N-linkedglycan structure, whereas a mixture of antibody molecules having glycansof various structures with various modifications for the structure ofthe non-reducing terminal or constituent saccharides or fragments ofsuch antibody molecules is provided (IV in FIG. 3A). Treatment of suchan antibody produced with an animal cell with hydrolase such as EndoScauses hydrolysis of the glycosidic bond at GlcNAcβ1-4GlcNAc in thechitobiose structure at the reducing terminal, providing antibodymolecules of single glycan structure having only (Fucα1,6)GlcNAc as N297glycan (referred to as “(Fucα1,6)GlcNAc-antibody”, see FIG. 2A) (FIG.3A) (step ii)).

For the enzyme for the hydrolysis reaction of N297 glycan, for example,EndoS or a variant enzyme retaining the hydrolysis activity may be used.

By reacting the (Fucα1,6)GlcNAc-antibody obtained in the abovehydrolysis reaction, as a glycan acceptor molecule, and an MSG- (MSG1-,MSG2-) or SG-type glycan donor molecule with use of transglycosidase(e.g., WO 2017010559) such as EndoS D233Q and EndoS D233Q/Q303Lvariants, an antibody of the above-described structure including MSG-(MSG1-, MSG2-) or SG type N297 glycan (see FIG. 2B) can be obtained(FIG. 3B) (step iii)-1, iii)-2).

If the number of conjugated drug molecules per antibody molecule, m², inthe antibody-drug conjugate is 1, a glycan donor molecule having MSG,MSG1, or MSG2 as glycan is employed. For such glycan, commerciallyavailable monosialo-Asn free (1S2G/1G2S-10NC-Asn, GlyTech, Inc.,hereinafter, referred to as “(MSG-)Asn”) as a raw material may beseparated in accordance with a method described in Example 56 to obtain(MSG-)Asn1 or (MSG2-)Asn, which may be employed, or a mixture of themmay be employed without separation.

If the number of conjugated drug molecules per antibody molecule, m², inthe antibody-drug conjugate is 2, a glycan donor molecule including SG(10) as glycan is used for the transglycosylation reaction. For such SG(10) glycan, for example, that obtained from SGP through hydrolysis orthe like may be used, or SG (10) glycan such as commercially availabledisialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) may be used.

MSG- (MSG1-, MSG2-) or SG-type glycan included in the donor molecule hasa PEG linker having an azide group (N₃-L(PEG)) at the 2-position of asialic acid therein. To introduce a PEG linker having an azide group(N₃-L(PEG)) to the 2-position of a sialic acid, a reaction known in thefield of synthetic organic chemistry (e.g., condensation reaction) maybe used for MSG (MSG (9)), MSG1, or MSG2, or disialooctasaccharide (SG(10)) and the PEG linker having an azide group (N₃-L(PEG))N₃—(CH₂CH₂—O)n₅-CH₂CH₂—NH₂, wherein n₅ is an integer of 2 to 10, andpreferably represents an integer of 2 to 5. Specifically, carboxylicacid at the 2-position of a sialic acid and the amino group at the rightend of N₃—(CH₂CH₂—O)n₅-CH₂CH₂—NH₂ undergo condensation reaction to forman amide bond.

Alternatively, MSG- (MSG1-, MSG2-) or SG-type glycan may be obtained byintroducing a PEG linker having an azide group(N₃—(CH₂CH₂—O)n₅-CH₂CH₂—NH₂) to carboxylic acid at the 2-position of asialic acid of a raw material such as (MSG1-)Asn, (MSG2-)Asn, and(SG-)Asn (GlyTech, Inc.) with an α-amino group optionally protected ormodified, and to carboxylic acid of the Asn with use of condensationreaction, and utilizing hydrolase such as EndoM and EndoRp (iii)-2).Examples of protective groups for α-amino groups may include, but notlimited to, an acetyl (Ac) group, a t-butoxycarbonyl (Boc) group, abenzoyl (Bz) group, a benzyl (Bzl) group, a carbobenzoxy (Cbz) group,and a 9-fluorenylmethoxycarbonyl (Fmoc) group. The protective group forα-amino groups is preferably an Fmoc group.

Examples of modifying groups for α-amino groups include modifying groupsthat enhance solubility in water with a hydroxyacetyl group, a PEGstructure, or the like.

An α-amino group of (MSG1-)Asn, (MSG-2)Asn, or (SG-)Asn is preferablyprotected with any of the protective groups. If an α-amino group isprotected with a protective group (e.g., an Fmoc group), the protectivegroup may be removed, as necessary, after introduction of a PEG linkerhaving an azide group and before causing action of hydrolase.

It is preferred to use an activated form such as an oxazolinated formformed by treatment with2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium-chloride for GlcNAc at thereducing terminal of MSG (MSG1, MSG2) or SG-type glycan included in themolecule.

Various enzymes for use in transglycosylation reaction(transglycosidase) may be employed that have activity of transferringcomplex glycan to N297 glycan; however, EndoS D233Q, a modified productfor which hydrolysis reaction is suppressed by substituting Asp at the233-position of EndoS with Gln, is a preferred transglycosidase.Transglycosylation reaction using EndoS D233Q is described, for example,in WO 2013/120066. Alternatively, a modified enzyme such as EndoSD233Q/Q303L (WO 2017010559), which is obtained by further adding amutation to EndoS D233Q, may be used.

The purification operation for the antibody after the glycan remodelingfor the antibody (glycohydrolysis and transglycosylation reaction) isintended to separate low-molecular-weight compounds and enzymes used forthe reaction, and gel filtration chromatography, ion-exchangechromatography, affinity chromatography, and so on are typically usedfor such purification, and additional purification with a hydroxyapatitecolumn may be further carried out. That is, the present inventionprovides a method for producing a drug-conjugate, the method including,subsequent to the step of purifying an intermediate from reactionsolution after glycohydrolysis of an antibody, the additional step ofpurifying with a hydroxyapatite column. According to an example ofreports on glycan remodeling (J. Am. Chem. Soc. 2012, 134, 12308-12318.,Angew. Chem. Int. Ed. 2016, 55, 2361-2367), reaction solution aftertreatment of an antibody with hydrolase is purified only with a ProteinA column (affinity chromatography column); however, this purificationmethod has been proved to be incapable of completely removing hydrolase(e.g., EndoS), and affect the subsequent transglycosylation reactionbecause of the residual enzyme. In view of such a result, examinationwas made on purification methods to find that when purification ofreaction solution after treatment of an antibody with hydrolase wascarried out using a Protein A column and a hydroxyapatite column (CHTcolumn, Bio-Rad Laboratories, Inc.) in the order presented, the reactionefficiency of the subsequent glycosylation reaction was enhanced,without the influence of a residual enzyme.

The antibody-drug conjugate of the present invention is the mostpreferably one antibody-drug conjugate selected from the followinggroup:

In each of the structural formulas above,

m² represents 1 or 2 (preferably, m² is 1),

antibody Ab is an IgG antibody (preferably, IgG1, IgG2, or IgG4, morepreferably, IgG1), or a functional fragment of the antibody,

N297 glycan represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and amixture of them, and N297-(Fuc)SG (preferably, N297-(Fuc)MSG1),

L(PEG) represents —NH—CH₂CH₂—(O—CH₂CH₂)₃—*, wherein the amino group atthe left end represents amide-bonding to carboxylic acid at the2-position of a sialic acid at the non-reducing terminal of each oreither one of the 1-3 and 1-6 branched chains (preferably, the 1-3branched chain) of β-Man in N297 glycan, and the asterisk representsbonding to a nitrogen atom at the 1- or 3-position of the triazole ringof Lb in linker L.

Although structures with two or four units (m²=1 or 2) of “—(N297glycan)-L-D” in each of which N297 glycan bonds to the nitrogen atom atthe 1-position of the triazole ring of Lb in L in one conjugate molecule(“(N297 glycan)-(N1Lb)L-D”) or structures with two or four units (m²=1or 2) of “—(N297 glycan)-L-D” in each of which N297 glycan bonds to thenitrogen atom at the 3-position of the triazole ring of Lb in L in oneconjugate molecule (“(N297 glycan)-(N3Lb)L-D”) are illustrated as themost preferred antibody-drug conjugate for convenience, antibody-drugconjugates having both “(N297 glycan)-(N1Lb)L-D” (if m²=1, then oneunit, if m²=2, then one, two, or three units) and “(N297glycan)-(N3Lb)L-D” (if m²=1, then one unit, if m²=2, then three, two, orone unit) in one conjugate molecule are also included. In other words,either one of “(N297 glycan)-(N1Lb)L-D” and “(N297 glycan)-(N3Lb)L-D”exists or both of them coexist in one conjugate molecule.

Further, Ab is preferably an anti-CLDN6 antibody, an anti-CLDN6/CLDN9antibody, an anti-HER2 antibody, an anti-DLL3 antibody, an anti-FAPantibody, an anti-CDH11 antibody, an anti-A33 antibody, an anti-CanAgantibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56antibody, an anti-CD70 antibody, an anti-CD98 antibody, an anti-TROP2antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2antibody, an anti-FGFR2 antibody, an anti-G250 antibody, an anti-MUC1antibody, an anti-GPNMB antibody, an anti-integrin antibody, ananti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4antibody, an anti-mesothelin antibody, an anti-EGFR antibody, or ananti-DR5 antibody, more preferably an anti-CLDN6 antibody, ananti-CLDN6/CLDN9 antibody, an anti-HER2 antibody, an anti-CD98 antibody,or an anti-TROP2 antibody, and even more preferably the anti-CLDN6antibody (e.g., Example 106, 107, 108, 109) or anti-HER2 antibody (e.g.,trastuzumab, a trastuzumab variant).

The antibody-drug conjugate of the present invention and the anti-CLDN6antibody- or anti-HER2 antibody-drug conjugate of the present inventionexhibit strong tumor activity (in vivo antitumor activity, in vitroanticellular activity) and satisfactory in vivo kinetics and physicalproperty, and have high safety, and hence are useful as apharmaceutical.

There may exist stereoisomers, optical isomers due to an asymmetriccarbon atom, geometric isomers, tautomers, or optical isomers such asd-forms, 1-forms and atropisomers for the antibody-drug conjugate of thepresent invention, and a free drug or production intermediate of theantibody-drug conjugate, and these isomers, optical isomers, andmixtures of them are all included in the present invention. PBDderivative (V) or (VI) of the present invention has an asymmetric carbonat the 11′-position, and thus there exist optical isomers. Herein, theseisomers and mixtures of these isomers are all represented by a singleformula, namely, general formula (V) or (VI). Accordingly, (V) or (VI)includes all the optical isomers and mixtures of the optical isomers atany ratio. The absolute steric configuration at the 11′-position of (V)or (VI) can be determined through X-ray crystal structure analysis orNMR such as a Mosher method for its crystalline product or intermediate,or a derivative thereof. Then, the absolute steric configuration may bedetermined by using a crystalline product or intermediate derivatizedwith a reagent having an asymmetric center whose steric configuration isknown. As desired, stereoisomers of the synthesized compound accordingto the present invention may be obtained by isolating with a commonoptical resolution method or separation method.

The number of conjugated drug molecules per antibody molecule is animportant factor having influence on efficacy and safety for theantibody-drug conjugate of the present invention. Antibody-drugconjugates are produced with reaction conditions, such as the amounts ofraw materials and reagents to be reacted, specified so as to give aconstant number of conjugated drug molecules, but, in contrast tochemical reaction of low-molecular-weight compounds, a mixture withdifferent numbers of conjugated drug molecules is typically obtained.Numbers of conjugated drug molecules per antibody molecule are specifiedas the average value, namely, the average number of conjugated drugmolecules (DAR: Drug to Antibody Ratio). The number ofpyrrolobenzodiazepine derivative molecules conjugated to an antibodymolecule is controllable, and 1 to 10 pyrrolobenzodiazepine derivativemolecules can be conjugated as the average number of conjugated drugmolecules per antibody molecule (DAR), but preferably the number is oneto eight, and more preferably one to five.

If the antibody bonds via a remodeled glycan of the antibody to L in theantibody-drug conjugate of the present invention, the number ofconjugated drug molecules per antibody molecule in the antibody-drugconjugate, m², is an integer of 1 or 2. If the glycan is N297 glycan andthe glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture ofN297-(Fuc)MSG1 and N297-(Fuc)MSG2, m² is 1, and DAR is in the range of 1to 3 (preferably, in the range of 1.0 to 2.5, more preferably, in therange of 1.2 to 2.2). If the N297 glycan is N297-(Fuc)SG, m² is 2, andDAR is in the range of 3 to 5 (preferably, in the range of 3.2 to 4.8,more preferably, in the range of 3.5 to 4.2).

Those skilled in the art could engineer the reaction method to conjugatea required number of drug molecules to each antibody molecule on thebasis of the description in Examples herein, and obtain an antibody witha controlled number of conjugated pyrrolobenzodiazepine derivativemolecules.

The antibody-drug conjugate, free drug, or production intermediate ofthe present invention may absorb moisture, allow adhesion of adsorbedwater, or become a hydrate when being left to stand in the atmosphere orrecrystallized, and such compounds and salts containing water are alsoincluded in the present invention.

The antibody-drug conjugate, free drug, or production intermediate ofthe present invention may be converted into a pharmaceuticallyacceptable salt, as desired, if having a basic group such as an aminogroup. Examples of such salts may include, but not limited to, hydrogenhalide salts such as hydrochlorides and hydroiodides; inorganic acidsalts such as nitrates, perchlorates, sulfates, and phosphates; loweralkanesulfonates such as methanesulfonates, trifluoromethanesulfonates,and ethanesulfonates; arylsufonates such as benzenesulfonates andp-toluenesulfonates; organic acid salts such as formates, acetates,malates, fumarates, succinates, citrates, tartrates, oxalates, andmaleates; and amino acid salts such as ornithinates, glutamates, andaspartates.

If the antibody-drug conjugate, free drug, or production intermediate ofthe present invention has an acidic group such as a carboxy group, abase addition salt can be generally formed. Examples of pharmaceuticalacceptable salts may include, but not limited to, alkali metal saltssuch as sodium salts, potassium salts, and lithium salts; alkali earthmetal salts such as calcium salts and magnesium salts; inorganic saltssuch as ammonium salts; and organic amine salts such as dibenzylaminesalts, morpholine salts, phenylglycine alkyl ester salts,ethylenediamine salts, N-methylglucamates, diethylamine salts,triethylamine salts, cyclohexylamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts, diethanolamine salts,N-benzyl-N-(2-phenylethoxy)amine salts, piperazine salts,tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts.

The antibody-drug conjugate, free drug, or production intermediate ofthe present invention may exist as a hydrate, for example, by absorbingmoisture in the air. The solvate of the present invention is not limitedto a particular solvate and may be any pharmaceutically acceptablesolvate, and specifically hydrates, ethanol solvates, 2-propanolsolvates, and so on are preferred. The antibody-drug conjugate, freedrug, or production intermediate of the present invention may be itsN-oxide form if a nitrogen atom is present therein, and these solvatesand N-oxide forms are included in the scope of the present invention.

The present invention includes compounds labeled with variousradioactive or nonradioactive isotopes. The antibody-drug conjugate,free drug, or production intermediate of the present invention maycontain one or more constituent atoms with non-natural ratios of atomicisotopes. Examples of atomic isotopes may include, but not limited to,deuterium (²H), tritium (³H), iodine-125 (¹²⁵I), and carbon-14 (¹⁴C).The compound of the present invention may be radiolabeled with aradioactive isotope such as tritium (³H), iodine-125 (¹²⁵I), andcarbon-14 (¹⁴C). The radiolabeled compound is useful as a therapeutic orprophylactic agent, a reagent for research such as an assay reagent, anda diagnostic agent such as a diagnostic agent for in vivo imaging.Isotopic variants of the antibody-drug conjugate of the presentinvention are all included in the scope of the present invention,regardless of whether they are radioactive or not.

[Production Methods]

Next, representative methods for producing the antibody-drug conjugateof the present invention and free drugs or production intermediatesthereof will be described. In the following, compound numbers shown inreaction formulas are used to identify compounds from each other.Specifically, reference in the form of “compound of formula (1)”,“compound (1)”, and so on will be made. Compounds with the other numberswill be indicated in the same manner.

1. Production Method 1

Compound (1) of the present invention may be produced in accordance withscheme A to scheme Q described in the following.

Scheme A to scheme M are each a method for producing a productionintermediate for the antibody-drug conjugate of the present invention.

Scheme N to scheme Q are each a method for producing a free drug of thepresent invention.

In each step in scheme A to scheme Q below, a desired reaction may becarried out by using a known technique of organic chemistry.

Solvent to be used in reaction of each step in scheme A to scheme Qbelow is not limited to a particular solvent and may be any solvent thatdissolves starting raw materials to some degree without inhibiting thereaction or having adverse effect on the reaction.

In each step in scheme A to scheme Q below, reaction temperature dependson solvent, starting raw materials, reagents, and so on, and reactiontime depends on solvent, starting raw materials, reagents, reactiontemperature, and so on.

In each step in scheme A to scheme Q below, a targeted compound iscollected by using a conventional method from a reaction mixture afterthe completion of reaction. For example, a reaction mixture isappropriately neutralized; if any insoluble matter is present theinsoluble matter is removed through filtration; an organic solventimmiscible with water, such as ethyl acetate, is then added to theresultant; an organic layer containing the targeted compound isseparated and washed with water or the like, and dried over anhydrousmagnesium sulfate, anhydrous sodium sulfate, or the like; and theresultant is filtered and the solvent is then distilled off to affordthe targeted product. The targeted product obtained may be subjected toseparation/purification, as necessary, by appropriately combiningconventional methods, for example, typical methods conventionally usedfor separation/purification of organic compounds such asrecrystallization, reprecipitation, and chromatography (e.g.,appropriately combining adsorption column chromatography methods with acarrier such as silica gel, alumina, a Florisil of magnesium-silica geltype, and S03H-silica (produced by FUJI SILYSIA CHEMICAL LTD.); methodswith a synthesized adsorbent such as partition column chromatographywith a carrier such as Sephadex LH-20 (produced by Pharmacia), AmberliteXAD-11 (produced by Rohm and Haas Company), and DIAION HP-20 (producedby Mitsubishi Chemical Corporation); methods using ion-exchangechromatography; normal phase/reversed-phase column chromatographymethods (preferably, high performance liquid chromatography) with silicagel or alkylated silica gel, and eluting with an appropriate eluent). Inthe case of a targeted compound insoluble in solvent, a crude product ofsolid obtained may be washed with solvent and purified. A targetedcompound in each step may be used for the subsequent reaction withoutpurification.

In each step in scheme A to scheme Q below, J, La′, Lp′, B′, E, V, W,R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, l, n⁷, n⁶, and m¹ have thesame meanings as described above. (Lp′)′ represents any of dipeptideresidues of -VA-, -FG-, -PI-, -VCit-, -VK-, -(D-)PI-, -PL-, -(D-)VA-,and -GF-. If a hydroxy group or an amino group is present on asubstituent of R³, a protective group may be used for (R¹³)′, and ifthere is no protective group, (R¹³)′ represents R¹³. (R¹⁷)′ representseither a hydroxy group protected with a protective group such as atert-butyldimethylsilyloxy group, or R¹⁷.

PRO¹, PRO⁴, PRO⁶, PRO⁸, and PRO⁹ each represent a protective group foran amino group. Preferably, PRO¹, PRO⁴, PRO⁸, and PRO⁹ each are, forexample, an allyloxycarbonyl group, a 2,2,2-trichloroethyloxycarbonylgroup, a trimethylsilylethoxymethoxy group, a benzyloxycarbonyl group,or a 9-fluorenylmethyloxycarbonyl group. PRO⁶ is preferably, forexample, a 2-(trimethylsilyl)ethoxymethyl group or a methoxymethy group.

PRO², PRO³, PRO⁵, PRO⁷, PRO¹⁰, PRO¹¹, and PRO¹² each represent aprotective group used in the field of synthetic organic chemistry for ahydroxy group, a phenol group, and a carboxyl group. Preferably, PRO²,PRO³, PRO⁵, PRO⁷, PRO¹⁰, PRO¹¹, and PRO¹² are each an acetyl group, abenzyl group, a tert-butyldimethylsilyl (TBDMS) group, atriisopropylsilyl group, or a tert-butyl group.

X² represents a leaving group used in the field of synthetic organicchemistry. Preferably, X² is a chlorine atom, a bromine atom, an iodineatom, a methanesulfonyl group, or a p-toluenesulfonyl group.

R^(a) and R^(c) each represent a substituent bonding to a carboxylgroup, and is preferably, for example, a methyl group, an ethyl group, abenzyl group, or a tert-butyl group.

R^(b) represents a leaving group to form enol sulfonate, and ispreferably, for example, a trifluoromethanesulfonyl group.

Amino groups and hydroxy groups without explicit description onprotection in scheme A to scheme Q may be protected, as necessary, byusing a protective group. Deprotection may be carried out, as necessary,and protection may be followed by deprotection to replace with anotherprotective group.

Scheme A

The production method is a method for producing compound (12a), asynthesized intermediate needed for production of compound (1).

Step A-1 (1a)→(2a): Reduction Reaction

The step is carried out by treating compound (1a) with a reducing agent(e.g., lithium aluminium hydride, diborane, lithium borohydride, sodiumborohydride, a borane-tetrahydrofuran complex, or sodiumbis(2-methoxyethoxy)aluminum hydride) in solvent (diethyl ether,tetrahydrofuran (THF), dichloromethane, ethanol, or the like, or mixedsolvent thereof) at −78° C. to the boiling point of the solvent used forthe reaction, preferably at −78° C. to 50° C. The amount of moles of thereducing agent to be used is 1 mol to an excessive amount of moles,preferably 1 to 5 mol, relative to compound (1a). As necessary, a Lewisacid (e.g., lithium chloride, calcium chloride, tin chloride, atrifluoroborane-ether complex) is added to the reaction. The reactiontime is 1 minute to 60 hours, and preferably 5 minutes to 24 hours.

Step A-2 (2a)→(3a): Introduction of Protective Group (e.g.,Tert-Butyldimethylsilyl Group)

When PRO² is a TBDMS group, the step is carried out by reacting compound(2a) with a silylating reagent (e.g., tert-butyldimethylsilyl chloride,tert-butyldimethylsilyl trifluoromethanesulfonate) in solvent(dichloromethane, acetonitrile, tetrahydrafuran, N,N-dimethylformamide(DMF), or the like, or mixed solvent thereof) at −20° C. to 120° C.,preferably at 0° C. to 100° C. As necessary, a base (e.g., imidazole,pyridine, 2,6-lutidine, 4-dimethylaminopyridine, sodium hydride) isadded to the reaction. The amount of moles of the silylating agent to beused is 1 mol to an excessive amount of moles, preferably 1 to 5 mol,relative to compound (2a), and the amount of moles of the base to beused is 1 mol to an excessive amount of moles, preferably 1 to 5 mol,relative to compound (2a). The reaction time is 1 minute to 72 hours,and preferably 5 minutes to 24 hours.

Step A-3 (3a)→(4a): Deprotection Reaction

When PRO¹ is a benzyloxycarbonyl group, the step is carried out bysubjecting compound (3a) to catalytic hydrogenation in solvent (ethanol,propanol, methanol, ethyl acetate, THF, 1,4-dioxane, or the like, ormixed solvent thereof) in the presence of a transition metal catalyst(e.g., palladium carbon) at 0° C. to the boiling point of the solventused for the reaction, preferably at 0° C. to 50° C. The step istypically carried out under the hydrogen atmosphere; however,cyclohexene, 1,4-cyclohexadiene, or the like may be used as a hydrogendonor, as necessary. The reaction time is 10 minutes to 100 hours, andpreferably 30 minutes to 72 hours.

Step A-4 (4a)→(5a): Condensation Reaction

The step is carried out by reacting compound (4a) and a carboxylic acid(compound (A)) in solvent (benzene, toluene, diethyl ether,dichloromethane, THF, DMF, water, or the like, or mixed solvent thereof)in the presence of a condensing agent such asN,N-dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoroborate, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride at−30° C. to the boiling point of the solvent used for the reaction,preferably at 0° C. to 50° C. The amount of moles of the carboxylic acid(compound (A)) to be used is 0.3 to 5 mol, preferably 0.4 to 2 mol, permole of compound (4a), and the amount of moles of the condensing agentto be used is 1 mol to an excessive amount of moles, preferably 1 to 5mol, per mole of compound (4a). As necessary, a base (e.g.,triethylamine, diisopropylethylamine, N-methylmorpholine,4-dimethylaminopyridine) and an additive (e.g., 1-hydroxybenzotriazole,1-hydroxy-7-azabenzotriazole) are added to the reaction. The amount ofmoles of the base to be used is a catalytic amount to an excessiveamount of moles, preferably 0.2 to 3 mol, per mole of compound (4a). Theamount of moles of the additive to be used is a catalytic amount to anexcessive amount, preferably 0.01 to 3 mol, per mole of compound (4a).The reaction time is 10 minutes to 72 hours, and preferably 30 minutesto 24 hours.

When the carboxylic acid (compound (A)) is to be converted into an acidhalide and subjected to condensation reaction, the step is carried outby reacting compound (4a) and the acid halide of the carboxylic acid(compound (A)) in solvent (benzene, toluene, diethyl ether,dichloromethane, tetrahydrofuran, dichloromethane, or the like, or mixedsolvent thereof) in the presence of a base (e.g., triethylamine,diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine) at−78° C. to the boiling point of the solvent used for the reaction,preferably at −50° C. to 100° C. The amount of moles of the acid halideto be used is 0.3 mol to 5 mol, preferably 0.4 mol to 2 mol, per mole ofcompound (4a), and the amount of moles of the base to be used is acatalytic amount to an excessive amount of moles, preferably 0.2 to 5mol, per mole of compound (4a). The reaction time is 10 minutes to 72hours, and preferably 30 minutes to 24 hours.

To prepare the acid halide compound of the carboxylic acid (compound(A)), the carboxylic acid (compound (A)) is treated with oxalylchloride, thionyl chloride, or the like in solvent (benzene, toluene,dichloromethane, dichloroethane, or the like, or mixed solvent thereof)at 0° C. to the boiling point of the solvent used for the reaction,preferably at 0° C. to 100° C. As necessary, a catalytic amount ofN,N-dimethylformamide or the like is added to the reaction. The amountof moles of oxalyl chloride or thionyl chloride to be used is 1 mol toan excessive amount of moles, preferably 1 to 10 mol, relative to thecarboxylic acid (compound (A)). The reaction time is 10 minutes to 72hours, and preferably 30 minutes to 24 hours.

Step A-5 (5a)→(6a): Reduction Reaction

The step is carried out by subjecting compound (5a) to catalytichydrogenation in solvent (ethanol, propanol, methanol, ethyl acetate,THF, 1,4-dioxane, DMF, or the like, or mixed solvent thereof) in thepresence of a transition metal catalyst (palladium carbon, nickel, orthe like) at 0° C. to the boiling point of the solvent used for thereaction, preferably at 0° C. to 50° C. The step is typically carriedout under the hydrogen atmosphere; however, cyclohexene,1,4-cyclohexadiene, hydrazine, or the like may be used as a hydrogendonor. The reaction time is 10 minutes to 72 hours, and preferably 30minutes to 24 hours.

Reduction of the nitro group may be carried out in the followingconditions.

The step is carried out by reacting compound (5a) and a reducing agent(e.g., iron, zinc, tin chloride) in solvent (ethanol, methanol, diethylether, ethyl acetate, or water, or mixed solvent thereof) at 0° C. tothe boiling point of the solvent used for the reaction, preferably at 0°C. to 90° C. As necessary, an acid (e.g., acetic acid, formic acid,ammonium chloride) is added to the reaction. The amount of moles of thereducing agent to be used is 1 mol to an excessive amount of moles,preferably 1 to 100 mol, per mole of compound (5a), and the amount ofmoles of the acid to be added is 1 mol to an excessive amount of molesper mole of compound (5a). The reaction time is 10 minutes to 72 hours,and preferably 30 minutes to 24 hours.

Step A-6 (6a)→(7a): Carbamatization Reaction

The step is carried out by reacting compound (6a) and triphosgene(isocyanating agent) in solvent (THF, dichloromethane, DMF, or the like,or mixed solvent thereof) at −30° C. to the boiling point of the solventused for the reaction, preferably at 0° C. to 50° C. to generate anisocyanate intermediate in the system, followed by treating with analcohol represented by general formula (B). As necessary, a base (e.g.,triethylamine, diisopropylethylamine, sodium carbonate, sodiumhydroxide) is added to the reaction. The amount of moles of triphosgene(isocyanating agent) to be used is 0.3 mol to an excessive amount ofmoles, preferably 0.35 to 3 mol, per mole of compound (6a), and theamount of moles of the base to be added is 0.5 to 5 mol per mole ofcompound (6a). The reaction time until the isocyanate intermediate hasformed is 10 minutes to 24 hours, and preferably 30 minutes to 1 hour.The reaction time for the reaction between the isocyanate intermediateand alcohol (B) is 10 minutes to 72 hours, and preferably 1 hour to 24hours.

Alcohol (B) to be used in the present step may be produced according toscheme L described later.

Step A-7 (7a)→(8a): Deprotection Reaction

When PRO² is a TBDMS group, the step is carried out by reacting compound(7a) and any of an acid (e.g., acetic acid), a desilylating reagent(e.g., hydrofluoric acid-pyridine, hydrofluoric acid-triethylamine, ahydrofluorate, hydrofluoric acid, tetra(n-butylammonium) fluoride), anda mixture of the acid and desilylating reagent in solvent(dichloromethane, chloroform, acetonitrile, methanol, ethanol, THF,water, or the like, or mixed solvent thereof) at −20° C. to 100° C.,preferably at 0° C. to 50° C. The amount of moles of the acid to be usedis 1 mol to an excessive amount of moles per mole of compound (7a), andthe amount of the acid or desilylating reagent to be used is 1 mol to anexcessive amount of moles, and preferably 1 to 10 mol, per mole ofcompound (7a). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

Step A-8 (8a)→(9a): Oxidation Reaction

The step is carried out by reacting compound (8a) and an oxidizing agent(e.g., a chlorosulfonium salt, a Dess-Martin reagent, tetrabutylammoniumruthenate, pyridinium chlorochromate, a nitroxy radical oxidationcatalyst) in solvent (acetone, dichloromethane, pyridine, or the like,or mixed solvent thereof) at −78° C. to the boiling point of the solventused for the reaction, preferably at −78° C. to 30° C. As necessary, abase (e.g., triethylamine, diisopropylethylamine, sodium hydrogencarbonate, sodium carbonate, sodium hydroxide) and a reoxidizing agent(e.g., N-methylmorpholine N-oxide, iodobenzene diacetate, sodiumhypochlorite) or additive (e.g., tetrabutylammonium bromide, potassiumbromide) is added to the reaction. The amount of moles of the oxidizingagent to be used is 0.005 mol to an excessive amount of moles,preferably 0.005 to 10 mol, per mole of compound (8a). The amount ofmoles of the base or reoxidizing agent to be added is 1 to 10 mol permole of compound (8a), and the amount of moles of the additive to beadded is 0.02 to 1 mol per mole of compound (8a). The reaction time is10 minutes to 72 hours, and preferably 30 minutes to 24 hours.

Step A-9 (9a)→(10a): Introduction of Protective Group

When (R¹⁷)′ is a tert-butyldimethylsilyloxy group, production is carriedout according to step A-2.

Step A-10 (10a)→(11a): Deprotection Reaction

When PRO³ is a triisopropylsilyl group, production is carried out bytreating compound (10a) with lithium acetate in solvent (DMF, water, orthe like, or a mixture thereof) at 0° C. to the boiling point of thesolvent used for the reaction, preferably at 0° C. to 50° C. The amountof moles of lithium acetate to be used is 1 mol to an excessive amountof moles, preferably 1 to 5 mol, per mole of compound (10a). Thereaction time is 10 minutes to 72 hours, and preferably 30 minutes to 24hours.

Step A-11 (11a)→(12a): Alkylation Reaction

The production is carried out by reacting compound (11a) and alkylatingagent (C) (e.g., 1,5-dibromopentane, 1,3-dibromopropane) in solvent(THF, DMF, or N,N-dimethylacetamide, or mixed solvent thereof) at −20°C. to the boiling point of the solvent used for the reaction, preferablyat 0° C. to the boiling point. As necessary, a base (e.g., potassiumcarbonate, cesium carbonate) is added to the reaction. The amount ofmoles of the alkylating agent to be used is 1 mol to an excessive amountof moles, preferably 1 to 10 mol, per mole of compound (11a), and theamount of moles of the base to be used is 0.4 mol to an excessive amountof moles, preferably 0.5 to 5 mol, per mole of compound (11a). Thereaction time is 1 minute to 60 hours, and preferably 5 minutes to 24hours.

Scheme B

The production method is a method for producing compound (10b), anintermediate needed for producing compound (1) in which R¹¹ and R¹² arecombined, together with the carbon atoms to which R¹¹ and R¹² are bound,to form a double bond and R¹⁴ and R¹⁵ are each hydrogen.

Step B-1 (1b)→(2b): Deprotection Reaction

When PRO⁷ is a triisopropylsilyl group, production is carried outaccording to step A-10 of scheme A.

When PRO⁷ is a benzyl group, production is carried out according to stepA-3 of scheme A.

Step B-2 (2b)→(3b): Alkylation Reaction

Production is carried out according to step A-11 of scheme A.

Step B-3 (3b)→(4b): Deprotection Reaction

When PRO⁵ is a TBDMS group, production is carried out according to stepA-7 of scheme A.

When PRO⁵ is an acetyl group, the step is carried out by reactingcompound (3b) and an appropriate base (e.g., potassium carbonate, sodiummethoxide, sodium hydroxide) in solvent (methanol, ethanol, THF, water,or the like, or mixed solvent thereof) at −20° C. to the boiling pointof the solvent used for the reaction, preferably at 0° C. to 50° C. Theamount of moles of the base to be used is a catalytic amount to anexcessive amount of moles, and preferably 0.1 to 10 mol. The reactiontime is 10 minutes to 72 hours, and preferably 30 minutes to 24 hours.

Step B-4 (4b)→(5b): Oxidation Reaction

The production is carried out according to step A-8 of scheme A.

Step B-5 (5b)→(6b): Enol Sulfonylation Reaction

When R^(b) is a trifluoromethanesulfonyl group, the step is carried outby reacting compound (5b) and trifluoromethanesulfonic anhydride or thelike in solvent (e.g., dichloromethane) at −78° C. to the boiling pointof the solvent used for the reaction, preferably at −78° C. to 30° C. Asnecessary, a base (e.g., 2,6-lutidine) is added to the reaction. Theamount of moles of trifluoromethanesulfonic anhydride to be used is 1mol to an excessive amount of moles, preferably 1 to 5 mol, per mole ofcompound (5b). The amount of moles of the base to be used is 1 mol to 10mol. The reaction time is 10 minutes to 24 hours, and preferably 30minutes to 6 hours.

Step B-6 (6b)→(7b): Cross Coupling Reaction (e.g., Suzuki-MiyauraReaction) with Transition Metal Catalyst

The step is carried out by using compound (6b) and an organic boroncompound (e.g., 4-methoxyphenylboronic acid) in solvent (ethanol,toluene, 1,4-dioxane, DMF, tetrahydrafuran, water, or the like, or mixedsolvent thereof) in the presence of a transition metal catalyst (e.g.,tetrakis(triphenylphosphine)palladium,dichlorobis(benzonitrile)palladium (II)) at 0° C. to the boiling pointof the solvent used for the reaction, preferably at 0° C. to 120° C. Asnecessary, a base (e.g., sodium carbonate, potassium carbonate, cesiumcarbonate, sodium hydrogen carbonate, sodium hydroxide) or an additive(e.g., silver oxide, triphenylarsine) is added to the reaction. Theamount of moles of the palladium catalyst to be used is 0.01 mol to 1mol, preferably 0.01 mol to 0.5 mol, per mole of compound (6b). Theamount of moles of the organic boron compound to be used is 1 mol to anexcessive amount of moles, preferably 1 mol to 10 mol, per mole ofcompound (6b), the amount of moles of the base to be used is 1 mol to 5mol per mole of compound (6b), and the amount of moles of the additiveto be used is 0.1 mol to 5 mol per mole of compound (6b). The reactiontime is 10 minutes to 72 hours, and preferably 30 minutes to 24 hours.

Step B-7 (7b)→(8b): Reduction Reaction

When PRO⁶ is a 2-(trimethylsilyl)ethoxymethyl group, for example, thestep is carried out by treating compound (7b) with a reducing agent(e.g., lithium borohydride, sodium borohydride) in solvent (diethylether, THF, dichloromethane, ethanol, or the like, or mixed solventthereof) at −78° C. to the boiling point of the solvent used for thereaction, preferably at −78° C. to 50° C. The amount of moles of thereducing agent to be used is 1 mol to an excessive amount of moles,preferably 1 to 30 mol, relative to 1 mol of compound (7b). The reactiontime is 1 minute to 24 hours, and preferably 5 minutes to 6 hours.Compound (8b) can be produced by adding silica gel to a solution(dichloromethane, ethanol, water, or mixed solvent thereof) of the crudeproduct obtained from the reduction reaction followed by treating withstirring. The silica gel to be used is in an excessive amount relativeto compound (7b). The treatment time is 12 hours to 150 hours, andpreferably 12 hours to 100 hours.

Step B-8 (8b)→(9b): Reduction of Imino Group

The step is carried out by treating compound (8b) with a reducing agent(e.g., sodium borohydride, cyanoborohydride, sodiumtriacetoxyborohydride, 2-picoline borane, pyridine borane) in solvent(THF, dichloromethane, N,N-dimethylforamide, or the like, or mixedsolvent thereof) at −78° C. to the boiling point of the solvent used forthe reaction, preferably at −78° C. to 50° C. The amount of moles of thereducing agent to be used is 1 mol to an excessive amount of moles,preferably 1 to 5 mol, relative to 1 mol of compound (8b). The reactiontime is 1 minute to 60 hours, and preferably 5 minutes to 24 hours.

Step B-9 (9b)→(10b): Introduction of Protective Group

When PRO⁸ is an allyloxycarbonyl group, the step is carried out byreacting compound (9b) and allyl chloroformate, diallyl dicarbonate, orthe like in solvent (benzene, toluene, pyridine, diethyl ether,dichloromethane, THF, 1,4-dioxane, water, or the like, or mixed solventthereof) at −30° C. to the boiling point of the solvent used for thereaction, preferably at 0° C. to 50° C. As necessary, a base (e.g.,triethylamine, diisopropylethylamine, pyridine, sodium carbonate,potassium carbonate, sodium hydroxide) is added to the reaction. Theamount of moles of allyl chloroformate to be used is 1 mol to anexcessive amount of moles, preferably 1 mol to 10 mol, per mole ofcompound (9b), and the amount of moles of the base to be used is 1 molto an excessive amount of moles, preferably 1 to 10 mol, per mole ofcompound (9b). The reaction time is 10 minutes to 72 hours, andpreferably 10 minutes to 48 hours.

When PRO⁸ is a 2,2,2-trichloroethoxycarbonyl group, the step is carriedout by reacting compound (9b) and 2,2,2-trichloroethyl chloroformate insolvent (benzene, toluene, pyridine, diethyl ether, dichloromethane,THF, 1,4-dioxane, water, or the like, or mixed solvent thereof) at −30°C. to the boiling point of the solvent used for the reaction, preferablyat 0° C. to 50° C. As necessary, a base (e.g., triethylamine,diisopropylethylamine, pyridine, sodium carbonate, sodium hydroxide) isadded to the reaction. The amount of moles of 2,2,2-trichloroethylchloroformate to be used is 1 mol to an excessive amount of moles,preferably 1 mol to 10 mol, per mole of compound (9b), and the amount ofmoles of the base to be used is 1 mol to an excessive amount, preferably1 to 10 mol, per mole of compound (9b). The reaction time is 10 minutesto 72 hours, and preferably 30 minutes to 48 hours.

Scheme C

The production method is a method for producing compound (14c), anintermediate needed for producing compound (1) in which R¹¹ and R¹² arecombined, together with the carbon atoms to which R¹¹ and R¹² are bond,to thereto form a double bond and R¹⁴ and R¹⁵ are each hydrogen.Compound (10b) may be produced by using the production method.

Step C-1 (1c)→(2c): Introduction of Protective Group

When PRO⁵ is an acetyl group, the step is carried out by reactingcompound (c) and an acetylating reagent (e.g., acetic anhydride, acetylchloride) in solvent (dichloromethane, DMF, pyridine, THF, 1,4-dioxane,or the like, or mixed solvent thereof) at −20° C. to the boiling pointof the solvent used for the reaction, preferably at 0° C. to 100° C. Asnecessary, a base (e.g., triethylamine, diisopropylethylamine, pyridine,4-dimethylaminopyridine) is added to the reaction. The amount of molesof the acetylating agent to be used is 1 mol to an excessive amount ofmoles, preferably 1 mol to 20 mol, per mole of compound (1c), and theamount of moles of the base to be used is a catalytic amount to anexcessive amount of moles, preferably 0.1 to 20 mol, per mole ofcompound (1c). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

When PRO⁵ is a TBDMS group, production is carried out according to stepA-2 of scheme A.

Step C-2 to Step C-5 and Step C-7 to Step C-14

Production in step C-2 is carried out according to step A-5 of scheme A,production in step C-3 is carried out according to step B-9 of scheme B,production in step C-4 is carried out according to step A-7 of scheme A,production in step C-5 is carried out according to step A-8 of scheme A,production in step C-7 is carried out according to step B-8 of scheme B,production in step C-8 is carried out according to step B-9 of scheme B,production in step C-9 is carried out according to step B-3 of scheme B,production in step C-10 is carried out according to step A-8 of schemeA, production in step C-11 is carried out according to step B-5 ofscheme B, production in step C-12 is carried out according to step B-6of scheme B, production in step C-13 is carried out according to stepA-10 of scheme A, and production in step C-14 is carried out accordingto step A-11 of scheme A.

Step C-6 (6c)→(7c): Deprotection Reaction

When PRO⁹ is a 2,2,2-trichloroethoxycarbonyl group, the step is carriedout by reacting compound (6c) and a metal reagent (e.g., zinc, zinc-leadalloy, cadmium, cadmium-lead) in solvent (THF, acetic acid, an aqueoussolution of ammonium acetate, water, or the like, or mixed solventthereof) at −20° C. to the boiling point of the solvent, preferably at0° C. to 40° C. The amount of moles of the metal reagent to be used is 1mol to an excessive amount of moles, preferably 1 to 10 mol, per mole ofcompound (6c). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

When PRO⁹ is an allyloxycarbonyl group, the step is carried out by usingcompound (6c), a palladium catalyst (e.g.,tetrakis(triphenylphosphine)palladium), and a scavenger for allyl groups(e.g., pyrrolidine, morpholine, barbituric acid) in solvent(dichloromethane, DMF, THF, or the like, or a mixture thereof) at 0° C.to the boiling point of the solvent used for the reaction, preferably at0° C. to 30° C. The amount of moles of the palladium catalyst to be usedis 0.005 mol to 1 mol, preferably 0.005 mol to 0.5 mol, per mole ofcompound (6c). The amount of moles of the scavenger for allyl groups tobe used is 1 mol to an excessive amount of moles, preferably 1 mol to 10mol. The reaction time is 10 minutes to 72 hours, and preferably 30minutes to 24 hours.

Scheme D

Compound (13c) may be produced by using the scheme.

Step D-1 to Step D-6, Step D-9, and Step D-10

Production in step D-1 is carried out according to step B-6 of scheme B,production in step D-2 is carried out according to step A-5 of scheme A,production in D-3 is carried out according to step B-9 of scheme B,production in step D-4 is carried out according to step A-7 of scheme A,production in step D-5 is carried out according to step A-8 of scheme A,production in step D-6 is carried out according to step C-6 of scheme C,production in step D-9 is carried out according to step B-8 of scheme B,and production in step D-10 is carried out according to step B-9 ofscheme B.

Step D-7 and Step D-8

Alternatively, compound (7d) may be produced according to step D-7,which is the same as step B-6 of scheme B, and step D-8, which is thesame as step B-7 of scheme B.

Scheme E

Scheme E is a method for producing compound (4e) by bonding compounds(11a) and (12a) produced in scheme A and compounds (10b) and (14c)produced in scheme B or scheme C.

Step E-1

The step is a step of producing compound (1e) through coupling reactionof compound (11a) produced in scheme A and compound (10b) produced inscheme B.

Production is carried out by subjecting compound (11a) to couplingreaction with compound (10b) in solvent (THF, DMF,N,N-dimethylacetamide, or mixed solvent thereof) in the presence of abase (e.g., potassium carbonate, cesium carbonate) at −20° C. to theboiling point of the solvent used for the reaction, preferably at 0° C.to 50° C. The amount of moles of compound (10b) to be used is 1 mol toan excessive amount of moles, preferably 0.7 to 1.5 mol, relative to 0.5mol of compound (11a). The amount of moles of the base to be used is 1mol to 5 mol relative to 0.5 mol of compound (11a). The reaction time is1 minute to 60 hours, and preferably 5 minutes to 24 hours.

Step E-2

Alternatively, compound (1e) may be produced by subjecting compound(12a) produced in scheme A and compound (14c) produced in scheme C tocoupling reaction as in step E-1.

Step E-3

Production in step E-3 is carried out according to step A-7 of scheme A.

Step E-4

The step is a step of producing compound (4e), when the protectivegroups PRO⁴ and PRO⁸ in compound (2e) are the same, by subjectingcompound (2e) to deprotection reaction as in step C-6 of scheme C.

When the protective groups PRO⁴ and PRO⁸ in compound (2e) are different,compound (4e) can be produced by stepwise deprotection reaction throughstep E-5 and step E-6.

Production in step E-5 and step E-6 is carried out according to step C-6of scheme C.

Scheme F

Alternatively, compound (4e) may be produced from intermediate compound(10f) in the synthesis method. The production method represents a methodfor producing compound (10f) and compound (4e).

Step F-1 to Step F-10, and Step F-15

Production in step F-1 is carried out according to step A-2 of scheme A,production in step F-2 is carried out according to step B-3 of scheme B,production in step F-3 is carried out according to step A-8 of scheme A,production in step F-4 is carried out according to step B-5 of scheme B,production in step F-5 is carried out according to step B-6 of scheme B,production in step F-6 is carried out according to construction methodA-2 of scheme A, production in step F-7 is carried out according to stepA-10 of scheme A, production in step F-8 is carried out according tostep A-11 of scheme A, production in step F-9 is carried out accordingto step E-1 of scheme E, production in step F-10 is carried outaccording to step E-1 of scheme E, and production in step F-15 iscarried out according to step B-8 of scheme B.

Step F-11

When PRO¹⁰ and the protective group for the hydroxy group in (R¹⁷)′ areeach a TBDMS group, production is carried out according to step A-7 ofscheme A.

Step F-12

The step is a step of producing compound (10f), when the protectivegroups PRO⁴ and PRO⁹ in compound (9f) are the same, by subjectingcompound (9f) to deprotection reaction as in step C-6 of scheme C.

Step F-13 and Step F-14

When the protective groups PRO⁴ and PRO⁹ in compound (9f) are different,compound (10f) can be produced in stepwise deprotection reaction throughstep F-13 and step F-14. Production in step F-13 and step F-14 iscarried out according to step C-6 of scheme C.

Scheme G

The production method is a method for producing compound (1 Ig), anintermediate for producing compound (1) in which R¹¹ represents ahydrogen atom, R¹² and R¹³ are combined to form a spiro ring, and R¹⁴and R¹⁵ each represent a hydrogen atom.

Steps G-1 and G-2, and Steps G-5 to G-11

Production in step G-1 is carried out according to step A-4 of scheme A,production in step G-2 is carried out according to step A-5 of scheme A,production in step G-5 is carried out according to step A-11 of schemeA, production in step G-6 is carried out according to step B-7 of schemeB, production in step G-7 is carried out according to step B-8 of schemeB, production in step G-8 is carried out according to step B-9 of schemeB, production in step G-9 is carried out according to step E-1 of schemeE, production in step G-10 is carried out according to step A-7 ofscheme A, and production in step G-1 is carried out according to stepC-6 of scheme C.

Step G-3: Introduction of Protective Group

The step is carried out by reacting compound (2g) and a chloromethoxyether-based reagent (e.g., 2-(chloromethoxy)ethyltrimethylsilane,chloromethyl methyl ether, benzyl chloromethyl ether) in solvent (THF,DMF, dioxane, or the like, or mixed solvent thereof) at −78° C. to theboiling point of the solvent, preferably at 0° C. to 50° C. Asnecessary, a base (sodium hydride, n-butyl lithium, hexamethyldisilazanelithium) is added to the reaction. The amount of moles of the reagent tobe used is 1 mol to an excessive amount of moles, preferably 1 to 5 mol,per mole of compound (2g). The amount of moles of the base to be used is1 mol to an excessive amount of moles, preferably 1 to 5 mol, per moleof compound (2g). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

Step G-4

When PRO⁷ is a benzyl group, production is carried out according to stepA-3 of scheme A.

When PRO⁷ is a triisopropylsilyl group, production is carried outaccording to step A-10 of scheme A.

Scheme H

The production method is a method for producing compound (9h), anintermediate for producing compound (1) in which R¹¹ represents ahydrogen atom, R¹² and R¹³ are combined to form a spiro ring, and R¹⁴and R¹⁵ are combined to represent an imine bond (C═N). In the productionmethod, the spiro ring formed by R¹² and R¹³ is synonymous with E, andhence represented by E.

Step H-1 to Step H-10

Production in step H-1 is carried out according to step A-4 of scheme A,production in step H-2 is carried out according to step A-1 of scheme A,production in step H-3 is carried out according to step C-1 of scheme C,production in step H-4 is carried out according to step A-4 of scheme A,production in step H-5 is carried out according to step A-5 of scheme A,production in step H-6 is carried out according to step B-9 of scheme B,production in step H-7 is carried out according to step A-6 of scheme A,production in step H1-8 is carried out according to step B-3 of schemeB, production in step H-9 is carried out according to step A-8 of schemeA, and production in step H1-10 is carried out according to step C-6 ofscheme C.

Scheme I

The production method is a method for producing compound (11i), anintermediate for producing compound (1) in which R¹¹ and R¹² arecombined to form a benzene ring, R¹³ is a single bond, and R¹⁴ and R¹⁵are combined to form imine.

Step I-1 to step I-4 h

Production in step I-1 is carried out according to step A-4 of scheme A,production in step I-2 is carried out according to step A-5 of scheme A,production in step I-3 is carried out according to step B-9 of scheme B,production in step I-4 is carried out according to step A-7 of scheme A,production in step I-5 is carried out according to step A-8 of scheme A,production in step I-6 is carried out according to step A-2 of scheme A,production in step I-7 is carried out according to step A-10 of schemeA, production in step I-8 is carried out according to step A-11 ofscheme A, production in step I-9 is carried out according to step E-1 ofscheme E, production in step I-10 is carried out according to step A-7of scheme A, and production in step I-11 is carried out according tostep C-6 of scheme C.

Scheme J

The production method is a method for producing compound (12j), anintermediate for producing compound (1) in which R¹² and R¹³ arecombined to form CH₂═, R¹¹ is hydrogen, and R¹⁴ and R¹⁵ are combined toform imine.

Step J-1

The step is a step of producing compound (2j) by subjecting compound(1j) to Wittig reaction.

Step J-2: Introduction of Protective Group

When PRO⁷ is a triisopropylsilyl group, the step is carried out byreacting compound (2j) and a silylating reagent (e.g., triisopropylsilylchloride, triisopropylsilyl triflate) in solvent (dichloromethane,acetonitrile, THF, DMF, or the like, or mixed solvent thereof) at −20°C. to 120° C., preferably at 0° C. to 100° C. As necessary, a base(e.g., imidazole, pyridine, 2,6-lutidine, 4-dimethylaminopyridine,sodium hydride) is added to the reaction. The amount of moles of thesilylating agent to be used is 1 mol to an excessive amount of moles,preferably 1 to 3 mol, per mole of compound (2a), and the amount ofmoles of the base to be used is 1 mol to an excessive amount of moles,preferably 1 to 5 mol, per mole of compound (2a). The reaction time is10 minutes to 72 hours, and preferably 30 minutes to 24 hours.

Step J-3 to Step J-11

Production in step J-3 is carried out according to step A-5 of scheme A,production in step J-4 is carried out according to step B-9 of scheme B,production in step J-5 is carried out according to step A-7 of scheme A,production in step J-6 is carried out according to step A-8 of scheme A,production in step J-7 is carried out according to step A-2 of scheme A,production in step J-8 is carried out according to step A-10 of schemeA, production in step J-9 is carried out according to step E-1 of schemeE, production in step J-10 is carried out according to step A-7 ofscheme A, and production in step J-11 is carried out according to stepC-6 of scheme C.

Scheme K

Scheme K is a method for producing compound (7k), an intermediate neededfor producing compound (1) in which R¹¹ and R¹² are combined, togetherwith the carbon atoms to which R¹¹ and R¹² are bond, to form a doublebond thereto, R¹³ is a hydroxymethyl group, and R¹⁴ and R¹⁵ togetherform imine.

Step K-1

The step is a step of producing compound (1k) by subjecting compound(6b) to carbonylation reaction.

Step K-2

The step is a step of producing compound (2k) by subjecting compound(1k) to aldehyde-selective reduction reaction.

Step K-3 to step K-7

Production in step K-3 is carried out according to step A-2 of scheme A,production in step K-4 is carried out according to step B-7 of scheme B,production in step K-5 is carried out according to step E-1 of scheme E,production in step K-6 is carried out according to step A-7 of scheme A,and production in step K-7 is carried out according to step C-6 ofscheme C.

Scheme L

Scheme L is a representative method for producing compound (B).

The peptide residues represented by general formula (Lp′)′ can beproduced through condensation reaction of amino acids.

PRO⁴ is protecting the N terminus of the peptide residues (Lp′)′ andPRO¹² is protecting the C terminus.

Step L-1

Production in step L-1 is carried out according to step B-9 of scheme B.

Step L-2: Deprotection Reaction

When PRO¹² is a tert-butyl group, the step is carried out by reactingcompound (21) and an acid (e.g., trifluoroacetic acid, p-toluenesulfonicacid, hydrochloric acid, acetic acid) in solvent (dichloromethane or thelike) at 0° C. to the boiling point of the solvent used for thereaction, preferably at 0° C. to 40° C. The amount of moles of the acidto be used is a catalytic amount to an excessive amount of moles permole of compound (2l). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

Step L-3 and Step L-4

Production in step L-3 is carried out according to step B-9 of scheme B,and production in step L-4 is carried out according to step A-4 ofscheme A.

Step L-5

Alternatively, compound (B) may be produced in step L-5 according tostep B-9 of scheme B.

Scheme M

Scheme M is a method for producing compound (2).

Compound (2) shown in the production method is synonymous with compound(1) such that R¹⁶ in the production intermediate of the presentinvention is J-La′-Lp′-NH—B′—CH₂—O(C═O)—*.

Compound (1m) shown in the production method represents compound (4e),(4f), (11g), (9h), (11i), (12j), (7k), or (8k) produced in any ofschemes E to K.

(PBD)′ shown in the production method represents:

and PBD represents:

wherein (PBD)′ may be protected with a substituent (e.g., a hydroxygroup) on R¹³ in PBD, and when lacking a protective group, (PBD)′ issynonymous with PBD (R¹³═(R¹³)′).

In the peptide residues represented by (Lp′)″_(t)-(Lp′)′ in theproduction method, a functional group (e.g., an amino group) on a sidechain of the amino acid residues represented by Lp′ may be protectedwith a protective group, and when a protective group is unsubstituted,(Lp′)″_(t)-(Lp′)′ is synonymous with Lp′.

(Lp′)′ represents an amino acid sequence of two amino acids as shownbelow, and when a functional group (an amino group, a hydroxy group) ispresent in a side chain, (Lp′)′ may be protected: -VA-, (D-)VA-, -FG-,-PI-, -VCit-, -VK-, -PL-, -(D-)P-I-, or -GF-.

(Lp′)″ represents an amino acid sequence of two to four amino acids asshown below, and when a functional group (an amino group, a hydroxygroup) is present in a side chain, (Lp′)″ may be protected:

-GG-, -EGG-, -DG-, -(D-)DG-, -EG-, -GGF-, -SG-, -KG-, -DGG-, -GGF-,-DDGG-, -KDGG-, or -GGFG-.

(La′)′ represents any one selected from the following group:—C(═O)—(CH₂CH₂)n⁶-C(═O),—C(═O)—(CH₂CH₂)n⁶-NH—C(═O)—(CH₂CH₂O)n⁷-CH₂CH₂—C(═O)—, —(CH₂)n⁸-O—C(═O)—,—(CH₂)n¹²-C(═O)—, and, —(CH₂CH₂)n¹³-C(═O)—NH—(CH₂CH₂O)n¹⁴-CH₂CH₂—C(═O)—

(La′)″ represents any one selected from the following group:—NH—(CH₂CH₂)n⁷-C(═O)— and —NH—(CH₂CH₂O)n⁷-CH₂—C(═O)—, and s and t eachindependently represent 0 or 1. For example, s and t are each 0 in stepM-1, s is 1 and t is 0 in step M-3, and s is 0 and t is 1 in step M-5.

(La′)′-(La′)″_(s) is synonymous with La′.

When having a protective group, (Lp′)″_(t)-(Lp′)′ is converted to Lp′through deprotection, and is synonymous with Lp′ when having noprotective group.

Lx shown in the production method represents a hydrogen atom or aleaving group (e.g., hydroxysuccinimide).

PBD or (PBD)′ in each of 1m, 9m, 10m, 11m, and compound (2) shown in theproduction method represents bonding at the asterisk (the N10′-position)to C(═O)— at the right end of —O—C(═O)—.

Step M-1

The step is a method of producing compound (11m) by subjecting compound(1m) produced in any of schemes E to K and compound (2m) to condensationreaction.

When Lx=H and compound (2m) is a carboxylic acid, compound (2m) can beproduced according to step A-4 of scheme A.

When Lx is a leaving group (e.g., hydroxysuccinimide, a p-nitrophenoxygroup), the step is carried out by reacting compound (1m) and compound(2m) in solvent (benzene, toluene, diethyl ether, dichloromethane, THF,DMF, methanol, water, or the like, or mixed solvent thereof) at −30° C.to the boiling point of the solvent used for the reaction, preferably at0° C. to 50° C. The amount of moles of compound (2m) to be used is 0.9mol to an excessive amount of moles, preferably 0.9 to 2 mol, per moleof compound (1m). As necessary, a base (e.g., triethylamine,N,N-diisopropylethylamine, N-methylmorpholine, 4-dimethylaminopyridine,diazabicycloundecene) is added to the reaction. The amount of moles ofthe base to be used is 1 mol to an excessive amount, preferably 1 to 5mol, per mole of compound (1m). The reaction time is 10 minutes to 72hours, and preferably 30 minutes to 36 hours.

Step M-2 to Step M-5 and Step M-8

Production in step M-2 is carried out according to step M-1, productionin step M-3 is carried out according to A-4 of scheme A, production instep M-4 is carried out according to step M-1, production in step M-5 iscarried out according to step A-4 of scheme A, and production in stepM-8 is carried out according to step A-4 of scheme A.

Step M-6

The step is a step of producing active ester intermediate (7m) bysubjecting compound (6m) to condensation reaction.

The step is carried out by reacting compound (6m) and hydroxysuccinimideor the like in solvent (benzene, toluene, diethyl ether,dichloromethane, THF, DMF, or the like, or mixed solvent thereof) in thepresence of a condensing agent such as N,N-dicyclohexylcarbodiimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at −30° C. to the boilingpoint of the solvent used for the reaction, preferably at 0° C. to 50°C. The amount of moles of the condensing agent to be used is 1 mol to anexcessive amount of moles, preferably 1 to 5 mol, per mole of compound(6m). The amount of moles of hydroxysuccinimide to be used is 1 mol toan excessive amount of moles, preferably 1 mol to 5 mol, per mole ofcompound (6m). The reaction time is 10 minutes to 72 hours, andpreferably 30 minutes to 24 hours.

Step M-7

The step is a step of producing compound (11m) by subjecting compound(1m) and compound (7m) to condensation reaction as in step M-1.

Step M-9

The step is a step of producing compound (10m) by subjecting compound(9m) to deprotection reaction. When PRO⁴ is a9-fluorenylmethyloxycarbonyl group, the step is carried out by reactingcompound (9m) and a base (e.g., 1,8-diazabicyclo[5.4.0]-7-undecene,piperidine) in solvent (THF, dichloromethane, DMF, or the like, or mixedsolvent thereof) at −20° C. to the boiling point of the solvent,preferably at 0° C. to 40° C. The amount of moles of the base to be usedis 1 mol to an excessive amount of moles, preferably 1 to 10 mol, permole of compound (9m). The reaction time is 1 minute to 72 hours, andpreferably 5 minutes to 24 hours.

Step M-10

The step is a step of producing compound (11m) by subjecting compound(10m) and compound (2m) or (4m) to condensation reaction as in step A-4of scheme A.

Step M-11

The step is a step of producing compound (2), when (Lp′)″_(t)-(Lp′)′ orPBD′ in compound (11m) has a protective group, by deprotecting compound(11m).

Production is carried out according to step B-3 of scheme B and step C-6of scheme C.

When (Lp′)′ or PBD′ has no protective group, step M-11 is omitted, andin this case compound (11m) is synonymous with compound (2).

Scheme N

Scheme N represents a synthesis method for a compound, as the free drugrepresented by (1) in which R¹¹ and R¹² are combined, together with thecarbon atoms to which R¹¹ and R¹² are bound, to form a double bond, R¹⁴and R¹⁵ are each hydrogen, and R¹⁶ and R¹⁷ are combined to form an iminebond.

Step N-1 to Step N-8

Production in step N-1 is carried out according to step B-9 of scheme B,production in step N-2 is carried out according to step A-7 of scheme A,production in step N-3 is carried out according to step A-8 of scheme A,production in step N-4 is carried out according to step A-2 of scheme A,production in step N-5 is carried out according to step A-10 of schemeA, production in step N-6 is carried out according to step A-11 ofscheme A, production in step N-7 is carried out according to step E-1 ofscheme E, and production in step N-8 is carried out according to stepE-1 of scheme E.

When (R¹³)′═R¹³, production is carried out according to step N-9 andstep N-10 shown in the following.

Step N-9

Production in step N-9 is carried out according to step A-7 of scheme A.

Step N-10

When the protective groups PRO⁴ and PRO⁸ are the same, production iscarried out according to step E-4 of scheme E. When the protectivegroups PRO⁴ and PRO⁸ are different, production is carried out accordingto steps E-5 and E-6 of scheme E.

When (R¹³)′ has a protective group, production is carried out accordingto step N-11 and step N-12 shown in the following.

Step N-11

Production is carried out according to step B-3 of scheme B.

Step N-12

When the protective groups PRO⁴ and PRO⁸ are the same, production iscarried out according to steps E-3 and E-4 of scheme E. When theprotective groups PRO⁴ and PRO⁸ are different, production is carried outaccording to steps E-3, E-5, and E-6 of scheme E.

Scheme O

Scheme O is a method for producing compound (6o), as the free drugrepresented by (1) in which R¹¹ and R¹² are combined, together with thecarbon atoms to which R¹¹ and R¹² are bound, to form a double bond, R¹⁴and R¹⁵ are combined to form an imine bond (C═N), and R¹⁶ and R¹⁷together form an imine bond (C═N).

Step O-1 to Step O-6

Production in step O-1 is carried out according to step E-1 of scheme E,production in step O-2 is carried out according to step B-3 of scheme B,production in step O-3 is carried out according to step A-8 of scheme A,production in step O-4 is carried out according to step B-5 of scheme B,production in step O-5 is carried out according to step B-6 of scheme B,and production in step O-6 is carried out according to step B-7 ofscheme B.

Scheme P

The production method is a method for producing a compound as the freedrug represented by (1) in which R¹¹ represents a hydrogen atom, R¹² andR¹³ are combined to form a spiro ring, R¹⁴ and R¹⁵ are combined to forman imine bond (C═N), and R¹⁶ and R¹⁷ together form an imine bond (C═N).In the production method, the spiro ring formed by R¹² and R¹³ incompound (4h) as a starting raw material is synonymous with E, and hencerepresented by E.

Step P-1 to Step P-4

Production in step P-1 is carried out according to step B-9 of scheme B,production in step P-2 is carried out according to step B-3 of scheme B,production in step P-3 is carried out according to step A-8 of scheme A,and production in step P-4 is carried out according to step C-6 ofscheme C.

Scheme Q

The production method is a method for producing a compound as the freedrug represented by (1) in which R¹¹, R¹⁴, and R¹⁵ each represent ahydrogen atom, R¹² and R¹³ are combined to form a spiro ring, and R¹⁶and R¹⁷ are combined to form an imine bond (C═N).

Step Q-1 to step Q-6

Production in step Q-1 is carried out according to step A-1 of scheme A,production in step Q-2 is carried out according to step A-8 of scheme A,production in step Q-3 is carried out according to step A-5 of scheme A,production in step Q-4 is carried out according to step E-1 of scheme E,production in step Q-5 is carried out according to step A-7 of scheme A,and production in step Q-6 is carried out according to step C-6 ofscheme C.

The protective group for optionally protected amino groups and hydroxygroups in the above description refers to a protective group cleavablewith a chemical method such as hydrogenolysis, hydrolysis, electrolysis,and photolysis, and represents a protective group commonly used insynthetic organic chemistry (e.g., see Protective groups in OrganicSynthesis, 3rd Edition, John Wiley & Sons, Inc. (1999)).

The “protective group” for optionally protected hydroxy groups (e.g., analkylcarbonyl group, a silyl group, or an aralkyl group), the“protective group” for optionally protected carboxy groups (e.g., aC₁-C₆ alkyl group or an aralkyl group), and the “protective group” foroptionally protected amino groups (e.g., an alkoxycarbonyl group) arenot limited to a particular protective group and may be any protectivegroup used for hydroxy groups, carboxy groups, and amino groups for usein the field of synthetic organic chemistry.

Steps requiring protection or deprotection are carried out according toany known method (e.g., a method described in “Protective groups inOrganic Synthesis” (by Theodora W. Greene, Peter G. M. Wuts, 1999,published by Wiley-Interscience Publication)).

Scheme R: Preparation of Antibody

A glycan-remodeled antibody may be produced by using a method asillustrated in FIGS. 3A and 3B, for example, according to a methoddescribed in WO 2013/120066 (see FIG. 50 ).

Step R-1: Hydrolysis of Glycosidic Bond at GlcNAcβ1-4GlcNAc ofChitobiose Structure at Reducing Terminal

The step is a step of preparing a glycan-truncated antibody by cleavingN-linked glycan bonding to asparagine at the 297-position of the aminoacid sequence of a targeted antibody (N297-linked glycan) with use of aknown enzymatic reaction.

A targeted antibody (20 mg/mL) in buffer solution (e.g., 50 mM phosphatebuffer solution) is subjected to hydrolysis reaction of the glycosidicbond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure at thereducing terminal with use of hydrolase such as the enzyme EndoS at 0°C. to 40° C. The reaction time is 10 minutes to 72 hours, and preferably1 hour to 6 hours. The amount of the wild-type enzyme EndoS to be usedis 0.1 to 10 mg, preferably 0.1 to 3 mg, to 100 mg of the antibody.After the completion of the reaction, purification with affinitychromatography and/or purification with a hydroxyapatite column, eachdescribed later, are/is carried out to produce a (Fucα1,6)GlcNAcantibody with the glycan hydrolyzed between GlcNAcβ1 and 4GlcNAc.Step R-2: Transglycosylation Reaction

The step is a step of producing a glycan-remodeled antibody by bondingthe (Fucα1,6)GlcNAc antibody to MSG- (MSG1-, MSG2-) or SG-type glycanoxazoline form (hereinafter, referred to as “azide glycan oxazolineform”) having a PEG linker including an azide group with use ofenzymatic reaction.

The glycan-truncated antibody in buffer solution (e.g., phosphate buffersolution) is subjected to transglycosylation reaction by reacting withan azide glycan oxazoline form in the presence of a catalytic amount oftransglycosidase such as EndoS (D233Q/Q303L) at 0° C. to 40° C. Thereaction time is 10 minutes to 72 hours, and preferably 1 hour to 6hours. The amount of the enzyme EndoS (D233Q/Q303L) to be used is 1 to10 mg, preferably 1 to 3 mg, to 100 mg of the antibody, and the amountof the azide glycan oxazoline form to be used is 2 equivalents to anexcessive equivalent, preferably 2 equivalents to 20 equivalents.

After the completion of the reaction, purification with affinitychromatography and purification with a hydroxyapatite column are carriedout to afford a purified glycan-remodeled antibody.

The azide glycan oxazoline form may be prepared according to methodsdescribed in Examples 55 to 57. By using a reaction known in the fieldof synthetic organic chemistry (e.g., condensation reaction),N₃—(CH₂CH₂—O)n₅-CH₂CH₂—NH₂, a PEG linker including an azide group(N₃-L(PEG)), may be introduced to MSG (MSG1, MSG2) ordisialooctasaccharide (Tokyo Chemical Industry Co., Ltd.). Specifically,carboxylic acid at the 2-position of a sialic acid and the amino groupat the right end of N₃—(CH₂CH₂—O)n₅-CH₂CH₂—NH₂ undergo condensationreaction to form an amide bond.

Examples of the condensing agent in using condensation reaction mayinclude, but not limited to, N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI),carbonyldiimidazole (CDI),2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (BOP),1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), and examples of the solvent for the reactionmay include, but not limited to, dichloromethane, DMF, THF, ethylacetate, and mixed solvent thereof.

The reaction temperature is typically −20° C. to 100° C. or the boilingpoint of the solvent, and preferably in the range of −5° C. to 50° C. Asnecessary, an organic base such as triethylamine, diisopropylethylamine,N-methylmorpholine, and 4-dimethylaminopyridine or an inorganic basesuch as potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogen carbonate may be added. Further, forexample, 1-hydroxybenzotriazole or N-hydroxysuccinimide may be added asa reaction accelerator.

MSG, MSG1, or MSG2 may be obtained by hydrolysis of the (MSG-)Asn orseparated/purified (MSG1-)Asn or (MSG2-)Asn (Example 56) with hydrolasesuch as EndoM.

Oxazolination may be prepared from GlcNAc at the reducing terminal ofMSG-(MSG1-, MSG2-) or SG-type glycan according to a known article (J.Org Chem., 2009, 74(5), 2210-2212. Helv. Chim. Acta, 2012, 95,1928-1936.).

In preparing the glycan-remodeled antibody, concentration of an aqueoussolution of an antibody, measurement of concentration, and bufferexchange may be carried out according to common operations A to C in thefollowing.

Common Operation A: Concentration of Aqueous Solution of Antibody

A solution of an antibody or antibody-drug conjugate was placed in acontainer of an Amicon Ultra (30,000 to 50,000 MWCO, MilliporeCorporation), and the solution of an antibody or antibody-drugconjugate, which is described later, was concentrated through acentrifugation operation (centrifugation at 2000 G to 4000 G for 5 to 20minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

Common Operation B: Measurement of Antibody Concentration

Measurement of antibody concentration was carried out by using a UVmeasurement apparatus (Nanodrop 1000, Thermo Fisher Scientific Inc.)according to a method specified by the manufacturer. Then, 280 nmabsorption coefficients, being different among antibodies (1.3 mL mg⁻¹cm⁻¹ to 1.8 mL mg⁻¹cm⁻¹), were used.

Common Operation C: Buffer Exchange for Antibody

A buffer solution (e.g., phosphate buffered saline (pH 6.0), phosphatebuffer (pH 6.0)) was added to an aqueous solution of an antibody, whichwas concentrated according to common operation A. This operation wascarried out several times, and the antibody concentration was thenmeasured by using common operation B, and adjusted to 10 mg/mL with abuffer solution (e.g., phosphate buffered saline (pH 6.0), phosphatebuffer (pH 6.0)).

Scheme S: Conjugation

The production method is a method for producing an antibody-drugconjugate by conjugating the above-described glycan-remodeled antibodyto production intermediate (2) through SPAAC (strain-promoted alkyneazide cycloaddition: J. AM. CHEM. SOC. 2004, 126, 15046-15047) reaction.In the formula, Ab represents the glycan-remodeled antibody.

SPAAC reaction proceeds by mixing a buffer solution (sodium acetatesolution, sodium phosphate, sodium borate solution, or the like, or amixture thereof) of antibody Ab and a solution dissolving compound (2)in an appropriate solvent (dimethyl sulfoxide (DMSO), dimethylformamide(DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), propyleneglycol (PG), or the like, or a mixture thereof).

The amount of moles of compound (2) to be used is 2 mol to an excessiveamount of moles, preferably 1 mol to 30 mol, per mole of the antibody,and the ratio of the organic solvent is preferably 1 to 200% v/v to thebuffer of the antibody. The reaction temperature is 0° C. to 37° C., andpreferably 10° C. to 25° C., and the reaction time is 1 to 150 hours,and preferably 6 hours to 100 hours. The pH in the reaction ispreferably 5 to 9.

Antibody-drug conjugate compounds (ADCs) can be identified from eachother through buffer exchange, purification, and measurement of antibodyconcentration and average number of conjugated drug molecules perantibody molecule according to common operations A to C described aboveand common operations D to F described later.

Common Operation D: Purification of Antibody-Drug Conjugate

An NAP-25 column was equilibrated with acetic acid buffer solution (10mM, pH 5.5; herein, referred to as ABS) containing commerciallyavailable sorbitol (5%). To this NAP-25 column, an aqueous reactionsolution of an antibody-drug conjugate (about 1.5 to 2.5 mL) wasapplied, and eluted with a buffer in an amount specified by themanufacturer to separate and collect an antibody fraction. The fractionseparated and collected was again applied to the NAP-25 column, and agel filtration purification operation to elute with a buffer wasrepeated twice or three times in total to afford the antibody-drugconjugate with an unbound drug-linker, dimethyl sulfoxide, and propyleneglycol removed. As necessary, the concentration of the solution of theantibody-drug conjugate was adjusted through common operations A to C.

Common Operation E: Measurement of Antibody Concentration ofAntibody-Drug Conjugate

The concentration of the conjugated drug in an antibody-drug conjugatecan be calculated by using the Lambert-Beer's law shown below.Expression (I) using the Lambert-Beer's law is as follows.

[Expression  1] $\begin{matrix}\begin{matrix}\underset{\_}{A_{280}} & = & \underset{\_}{ɛ_{280}\left( {L \cdot {mol}^{- 1} \cdot {cm}^{- 1}} \right)} & \cdot & \underset{\_}{C\left( {{mol}^{- 1} \cdot L^{- 1}} \right)} & \cdot & \underset{\_}{l({cm})} \\{Absorbance} & = & {{Molar}\mspace{14mu}{absorption}\mspace{14mu}{coefficient}} & \times & {Molarity} & \times & \begin{matrix}{Optical} \\{{path}\mspace{14mu}{length}}\end{matrix}\end{matrix} & {{Expression}\mspace{14mu}(I)}\end{matrix}$Here, A280 denotes absorbance of an aqueous solution of an antibody-drugconjugate at 280 nm, ε280 denotes the molar absorption coefficient of anantibody-drug conjugate at 280 nm, and C (mol·L⁻¹) denotes the molarityof an antibody-drug conjugate. From expression (I), the molarity of anantibody-drug conjugate, C (mol·L⁻¹), can be determined by usingexpression (II) below.

[Expression  2]                                     $\begin{matrix}{{C\left( {{mol}^{- 1} \cdot L^{- 1}} \right)} = \frac{A_{280}}{{ɛ_{280}\left( {L \cdot {mol}^{- 1} \cdot {cm}^{- 1}} \right)} \cdot {l({cm})}}} & {{Expression}\mspace{14mu}({II})}\end{matrix}$Further, the both sides are multiplied by the molar mass of theantibody-drug conjugate, MW (g·mol⁻¹), to determine the weightconcentration of the antibody-drug conjugate, C′ (mg·mL⁻¹) (expression(III)).

[Expression  3]                                     $\begin{matrix}{{C^{\prime}\left( {{mg} \cdot {mL}^{- 1}} \right)} = {{{{MW}\left( {g \cdot {mol}^{- 1}} \right)} \cdot {C\left( {{mol} \cdot L^{- 1}} \right)}} = \frac{A_{280} \times {MW}\mspace{14mu}\left( {g \cdot {mol}^{- 1}} \right)}{{ɛ_{280}\left( {L \cdot {mol}^{- 1} \cdot {cm}^{- 1}} \right)} \cdot {l({cm})}}}} & {{Expression}\mspace{14mu}({III})}\end{matrix}$

Values used for the expression and applied to Examples will bedescribed.

The absorbance A280 used was a measured value of UV absorbance of anaqueous solution of an antibody-drug conjugate at 280 nm. For molarmass, MW (g·mol⁻¹), an estimated value of the molecular weight of anantibody was calculated from the amino acid sequence of the antibody,and used as an approximate value of the molar mass of an antibody-drugconjugate. The optical path length, 1 (cm), used in measurement was 1cm.

The molar absorption coefficient, ε280, of the antibody-drug conjugatecan be determined by using expression (IV) below.

Here, ε_(Ab, 280) denotes the molar absorption coefficient of anantibody at 280 nm, and ε_(DL, 280) denotes the molar absorptioncoefficient of a drug at 280 nm.

By using a known calculation method (Protein Science, 1995, vol. 4,2411-2423), ε_(Ab, 280) can be estimated from the amino acid sequence ofan antibody. In Examples, the molar absorption coefficient oftrastuzumab used was ε_(Ab, 280)=215400 (calculated estimated value).The molar absorption coefficient of the CLDN6 antibody used wasε_(Ab, 280)=221340 (calculated estimated value), the molar absorptioncoefficient of the TROP2 antibody used was ε_(Ab, 280)=226400(calculated estimated value), the molar absorption coefficient of theCD98 antibody used was ε_(Ab, 280)=240400 (calculated estimated value),the molar absorption coefficient of the LPS antibody used wasε_(Ab, 280)=230300 (calculated estimated value), and the molarabsorption coefficient of the trastuzumab variant used wasε_(Ab, 280)=215057 (calculated estimated value).

ε_(DL, 280) was calculated for use from a measured value obtained ineach UV measurement. Specifically, the absorbance of a solutiondissolving a conjugate precursor (drug) with a certain molarity wasmeasured, and expression (I), the Lambert-Beer's law, was appliedthereto, and the resulting value was used.

Common Operation F: Measurement of Average Number of Conjugated DrugMolecules Per Antibody Molecule in Antibody-Drug Conjugate

The average number of conjugated drug molecules per antibody molecule inan antibody-drug conjugate can be determined through high-performanceliquid chromatography (HPLC) with the following method.

[F-1. Preparation of Sample for HPLC Analysis (Reduction ofAntibody-Drug Conjugate)]

A solution of an antibody-drug conjugate (about 1 mg/mL, 60 μL) is mixedwith an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). Themixture is incubated at 37° C. for 30 minutes to prepare a sample inwhich the disulfide bond between the L chain and H chain of theantibody-drug conjugate cleaved, and this sample is used for HPLCanalysis.

[F-2. HLPC Analysis]

HPLC analysis is carried out under the following conditions.

HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

Detector: Ultraviolet absorption spectrometer (measurement wavelength:280 nm, 329 nm)

Column: BEH Phenyl (2.1×50 mm, 1.7 μm, Waters Acquity)

Column temperature: 75° C.

Mobile phase A: 0.1% trifluoroacetic acid (TFA)-15% isopropyl alcoholaqueous solution

Mobile phase B: 0.075% TFA-15% isopropyl alcohol acetonitrile solution

Gradient program: 14%-36% (0 min to 15 min), 36%-80% (15 min to 17 min),80%-14% (17 min to 17.1 min), 14%-14% (17.1 min to 23 min)

Sample injection volume: 5 μL

[F-3. Data Analysis]

[F-3-1] An L chain with a conjugated drug molecule (L chain with oneconjugated drug molecule: L₁) and H chain with a conjugated drugmolecule(s) (H chain with one conjugated drug molecule: H₁, H chain withtwo conjugated drug molecules: H₂, H chain with three conjugated drugmolecules: H₃) have hydrophobicity increased in proportion to the numberof conjugated drug molecules and have longer retention time as comparedto the L chain (L₀) and H chain (H₀) of an antibody without anyconjugated drug molecule, and hence L₀, L₁, H₀, H₁, H₂, and H₃ areeluted in the presented order. While the order of L₁ and H₀ is inversedin some cases, H₀, which has no conjugated drug molecule, does notabsorb at a wavelength of 329 nm characteristic to drugs. Therefore, L₁and H₀ can be distinguished by checking absorption at a wavelength of329 nm. Through comparison of retention time between L₀ and H₀, eachpeak detected can be assigned to L₀, L₁, H₀, H₁, H₂, or H₃.[F-3-2] Since each drug-linker absorbs UV, peak area values arecorrected by using the following expression with the molar absorptioncoefficients of an L chain, H chain, and drug-linker according to thenumber of conjugated drug-linker molecules.

[Expression  5] $\begin{matrix}{{Corrected}\mspace{14mu} L\mspace{14mu}{chain}\mspace{14mu}{peak}} \\{{area}\mspace{14mu}({Li})}\end{matrix} = {{Peak}\mspace{14mu}{area} \times \frac{{Molar}\mspace{14mu}{absorption}\mspace{14mu}{coefficient}\mspace{14mu}{of}\mspace{14mu} L\mspace{14mu}{chain}}{\begin{matrix}{{Molar}\mspace{14mu}{absorption}} \\{{coefficient}\mspace{14mu}{of}\mspace{14mu} L\mspace{14mu}{chain}}\end{matrix} + {\begin{matrix}{{Number}\mspace{14mu}{of}} \\{conjugated} \\{{drug}\mspace{14mu}{molecules}}\end{matrix} \times \begin{matrix}{{Molar}\mspace{14mu}{absorption}\mspace{14mu}{coefficient}} \\{{of}\mspace{14mu}{drug}\text{-}{linker}}\end{matrix}}}}$ $\begin{matrix}{{Corrected}\mspace{14mu} H\mspace{14mu}{chain}\mspace{14mu}{peak}} \\{{area}\mspace{14mu}({Hi})}\end{matrix} = {{Peak}\mspace{14mu}{area} \times \frac{{Molar}\mspace{14mu}{absorption}\mspace{14mu}{coefficient}\mspace{14mu}{of}\mspace{14mu} H\mspace{14mu}{chain}}{\begin{matrix}{{Molar}\mspace{14mu}{absorption}} \\{{coefficient}\mspace{14mu}{of}\mspace{14mu} H\mspace{14mu}{chain}}\end{matrix} + {\begin{matrix}{{Number}\mspace{14mu}{of}} \\{conjugated} \\{{drug}\mspace{14mu}{molecules}}\end{matrix} \times \begin{matrix}{{Molar}\mspace{14mu}{absorption}\mspace{14mu}{coefficient}} \\{{of}\mspace{14mu}{drug}\text{-}{linker}}\end{matrix}}}}$Here, for the molar absorption coefficients (280 nm) of the L chain andH chain of each antibody, values estimated from the amino acid sequencesof the L chain and H chain of the antibody by using a known calculationmethod (Protein Science, 1995, vol. 4, 2411-2423) may be used. In thecase of trastuzumab, 26150 was used as the molar absorption coefficientof the L chain estimated from the amino acid sequence, and 81290 wasused as the molar absorption coefficient of the H chain estimated fromthe amino acid sequence. In the case of the CLDN6 antibody, similarly,33140 was used as the molar absorption coefficient of the L chain, and77280 was used as the molar absorption coefficient of the H chain; inthe case of the TROP2 antibody, 26210 was used as the molar absorptioncoefficient of the L chain, and 68990 was used as the molar absorptioncoefficient of the H chain; in the case of the CD98 antibody, 41680 wasused as the molar absorption coefficient of the L chain, and 78500 wasused as the molar absorption coefficient of the H chain; in the case ofthe LPS antibody, 31710 was used as the molar absorption coefficient ofthe L chain, and 77470 was used as the molar absorption coefficient ofthe H chain; in the case of the trastuzumab variant, 26251 was used asthe molar absorption coefficient of the L chain, and 81488 was used asthe molar absorption coefficient of the H chain; and the molarabsorption coefficient (280 nm) measured for compound (1), as aconjugate precursor, was used as the molar absorption coefficient (280nm) of each drug-linker.[F-3-3] The peak area ratio (%) of each chain to the total of correctedpeak areas is calculated by using the following expression.

[Expression  6]                                    ${L\mspace{14mu}{chain}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{ratio}} = {\frac{A_{Li}}{A_{L\; 0} + A_{L\; 1}} \times 100}$${H\mspace{14mu}{chain}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{ratio}} = {\frac{A_{Hi}}{A_{H\; 0} + A_{H\; 1} + A_{H\; 2} + A_{H\; 3}} \times 100}$

A_(Li), A_(Hi): L_(i), H_(i) Respective corrected peak areas

[F-3-4] The average number of conjugated drug molecules per antibodymolecule in an antibody-drug conjugate is calculated by using thefollowing expression.Average number of conjugated drug molecules=(L ₀ peak area ratio×0+L ₀peak area ratio×1+H ₀ peak area ratio×0+H ₁ peak area ratio×1+H ₂ peakarea ratio×2+H ₃ peak area ratio×3)/100×2<Medicine>

The antibody-drug conjugate of the present invention exhibits cellularcytotoxic activity to cancer cells, and hence may be used as a medicine,in particular, a therapeutic agent and/or prophylactic agent for cancer.

Examples of cancers to which the antibody-drug conjugate of the presentinvention is applied may include lung cancer (e.g., non-small cell lungcancer, small cell lung cancer), kidney cancer, urothelial cancer,colorectal cancer, prostate cancer, glioblastoma multiforme, ovariancancer (e.g., surface epithelial tumor, stromal tumor, germ cell tumor),pancreatic cancer, breast cancer, melanoma, liver cancer, bladdercancer, gastric cancer, esophageal cancer or the like, endometrialcancer, testicular cancer (seminoma, non-seminoma), uterine cervixcancer, placental choriocarcinoma, brain tumor, and head-and-neckcancer, and metastatic forms of them, but are not limited thereto aslong as cancer cells as a therapeutic target are expressing proteinrecognizable for the antibody in the antibody-drug conjugate.

The antibody-drug conjugate of the present invention can be preferablyadministered to mammals, and are more preferably administered to humans.

Substances used in a pharmaceutical composition containing theantibody-drug conjugate of the present invention may be suitablyselected and applied from formulation additives or the like that aregenerally used in the field in view of the dose or concentration foradministration.

The antibody-drug conjugate of the present invention may be administeredas a pharmaceutical composition containing one or more pharmaceuticallyapplicable components. For example, the pharmaceutical compositiontypically contains one or more pharmaceutical carriers (e.g., sterilizedliquid (including water and oil (petroleum oil and oil of animal origin,plant origin, or synthetic origin (such as peanut oil, soybean oil,mineral oil, and sesame oil)))). Water is a more typical carrier whenthe pharmaceutical composition above is intravenously administered.Saline solution, an aqueous dextrose solution, and an aqueous glycerolsolution can be also used as a liquid carrier, in particular, for aninjection solution. Suitable pharmaceutical vehicles are known in theart. If desired, the composition above may also contain a trace amountof a moisturizing agent, an emulsifying agent, or a pH buffering agent.Examples of suitable pharmaceutical carriers are disclosed in“Remington's Pharmaceutical Sciences” by E. W. Martin. The formulationscorrespond to the administration mode.

Various delivery systems are known and they may be used foradministering the antibody-drug conjugate of the present invention.Examples of the administration route may include, but not limited to,intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous routes. The administration may be made by injection orbolus injection, for example. According to a specific preferredembodiment, the administration of the above ligand-drug conjugate formis done by injection. Parenteral administration is a preferredadministration route.

According to a representative embodiment, the pharmaceutical compositionis prescribed, as a pharmaceutical composition suitable for intravenousadministration to humans, according to conventional procedures. Thecomposition for intravenous administration is typically a solution in asterile and isotonic aqueous buffer. If necessary, the medicine maycontain a solubilizing agent and a local anesthetic to alleviate pain atan injection site (e.g., lignocaine). Generally, the ingredients aboveare provided either individually as a dried lyophilized powder or ananhydrous concentrate contained in each container which is obtained bysealing in an ampoule or a sachet with indication of the amount of theactive agent, or as a mixture in a unit dosage form. When thepharmaceutical composition is to be administered by injection, it may beadministered from an injection bottle containing water or saline ofsterile pharmaceutical grade. When the medicine is administered byinjection, an ampoule of sterile water or saline for injection may beprovided so that the aforementioned ingredients are admixed with eachother before administration.

The pharmaceutical composition of the present invention may be apharmaceutical composition containing only the present antibody-drugconjugate, or a pharmaceutical composition containing the antibody-drugconjugate and at least one cancer treating agent other than theantibody-drug conjugate. The antibody-drug conjugate of the presentinvention may be administered in combination with other cancer treatingagents, and thereby the anti-cancer effect may be enhanced. Otheranti-cancer agents used for such purpose may be administered to anindividual simultaneously with, separately from, or subsequently to theantibody-drug conjugate, and may be administered while varying theadministration interval for each. Examples of such cancer treatingagents may include abraxane, carboplatin, cisplatin, gemcitabine,irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin,agents described in International Publication No. WO 2003/038043, LH-RHanalogues (e.g., leuprorelin, goserelin), estramustine phosphate,estrogen antagonists (e.g., tamoxifen, raloxifene), and aromataseinhibitors (e.g., anastrozole, letrozole, exemestane), but are notlimited thereto as long as they are agents having an antitumor activity.

The pharmaceutical composition can be formulated into a lyophilizationformulation or a liquid formulation as a formulation having the selectedcomposition and required purity. When formulated as a lyophilizationformulation, it may be a formulation containing suitable formulationadditives that are used in the art. Also for a liquid formulation, itmay be formulated as a liquid formulation containing various formulationadditives that are used in the art.

The composition and concentration of the pharmaceutical composition mayvary depending on the administration method. However, the antibody-drugconjugate contained in the pharmaceutical composition of the presentinvention can exhibit a pharmaceutical effect even at a small dosagewhen the antibody-drug conjugate has a higher affinity for an antigen,that is, a higher affinity (lower Kd value) in terms of the dissociationconstant (Kd value) for the antigen. Thus, for determining the dosage ofthe antibody-drug conjugate, the dosage may be set in view of thesituation relating to the affinity of the antibody-drug conjugate withthe antigen. When the antibody-drug conjugate of the present inventionis administered to a human, for example, about 0.001 to 100 mg/kg can beadministered once or administered in several portions with intervals of1 to 180 days.

The antibody of the present invention or a functional fragment of theantibody may be used as a medicine. In this case, the above descriptionof “antibody-drug conjugate” in the above chapter <Medicine> may beappropriately read as a description of the “antibody or functionalfragment of the antibody”.

Further, the free drug of the present invention (novel PBD derivativecompound), a salt of the free drug, and hydrates of them may be used asa medicine. In this case, the above description of “antibody-drugconjugate” in the above chapter <Medicine> may be appropriately read asa description of the “free drug (novel PBD derivative compound), a saltof the free drug, and hydrates of them”.

EXAMPLES

The present invention will be specifically described with reference toExamples shown below; however, the present invention is not limited toExamples. Examples should not be interpreted as limitation in any sense.Reagents, solvents, and starting materials without any descriptionherein can be readily obtained from commercially available sources ofsupply.

Reference Example 1: Trastuzumab-Tesirine

Step 1: Conjugation of Antibody and Drug-Linker

To a 5 mM solution of trastuzumab (Reference Example 3) inethylenediamine tetraacetate-phosphate buffered saline (pH 6.5) (9.91mg/mL, 0.70 mL), an aqueous solution of dipotassium phosphate (1.0 M,0.0112 mL) and an aqueous solution of tris(2-carboxyethyl)phosphinehydrochloride (10 mM, 0.0086 mL) were added at 20° C., and reacted at20° C. for 60 minutes and then at room temperature for 30 minutes. Asolution of tesirine (0.36 mg) synthesized with reference to aliterature (Med. Chem. Lett. 2016, 7, 983-987) in dimethylacetamide(0.0415 mL) was added to the reaction solution, and reacted at roomtemperature for 1 hour. An aqueous solution of N-acetylcysteine (100 mM,0.0024 mL) was added to the reaction solution, and reacted for 30minutes to terminate the reaction.

Purification operation: The solution was purified by using commonoperation D to afford 3.5 mL of a solution of the targeted compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.40 mg/mL, antibody yield: 4.90 mg (71%),average number of conjugated drug molecules per antibody molecule (n):2.0

Reference Example 2: Anti-CLDN6 (H1L1)-Tesirine

Step 1: Conjugation of Antibody and Drug-Linker

To a 5 mM solution of an anti-CLDN6 (H1L1) antibody in ethylenediaminetetraacetate-phosphate buffered saline (pH 6.5) (9.87 mg/mL, 0.45 mL),an aqueous solution of dipotassium phosphate (1.0 M, 0.0072 mL) and anaqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (10 mM,0.0041 mL) were added at 20° C., and reacted at 20° C. for 90 minutes. Asolution of tesirine (0.15 mg) synthesized with reference to aliterature (Med. Chem. Lett. 2016, 7, 983-987) in N,N-dimethylacetamide(0.0277 mL) was added to the reaction solution, and reacted at 20° C.for 1 hour. An aqueous solution of N-acetylcysteine (100 mM, 0.001 mL)was added to the reaction solution, and reacted for 30 minutes toterminate the reaction.

Purification operation: The solution was purified by using commonoperation D to afford 3.5 mL of a solution of the targeted compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.56 mg/mL, antibody yield: 3.90 mg (88%),average number of conjugated drug molecules per antibody molecule (n):2.1

Reference Example 3: Anti-HER2 Antibody Trastuzumab

The anti-HER2 antibody was produced with reference to U.S. Pat. No.5,821,337. The amino acid sequences of the light chain and heavy chainof trastuzumab are represented by SEQ ID NO: 64 and SEQ ID NO: 65,respectively.

Reference Example 4: Anti-LPS Antibody h #1G5-H1L1

The anti-LPS antibody was produced with reference to WO 2015/046505. Theamino acid sequences of the light chain and heavy chain of h #1G5-H1L1are represented by SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

Reference Example 5: Anti-TROP2 Antibody hRS7

The anti-TROP2 antibody was produced with reference to WO 2003/074566and WO 2015/098099 (Reference Example 1). The amino acid sequences ofthe light chain and heavy chain of hRS7 are represented by SEQ ID NO: 68and SEQ ID NO: 69, respectively.

Reference Example 6: Anti-CD98 Antibody hM23-H1L1

The anti-CD98 antibody was produced with reference to WO 2015/146132.The amino acid sequences of the light chain and heavy chain of hM23-H1L1are represented by SEQ ID NO: 70 and SEQ ID NO: 71, respectively.

Synthesis of Production Intermediate Example 1: Intermediate 1

Step 1: Benzyl(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate

To a solution of 5-benzyl 6-methyl(6S)-5-azaspiro[2.4]heptane-5,6-dicarboxylate (104 mmol, WO 2012087596)in THF (500 mL), lithium borohydride (4.30 g, 178 mmol) was added insmall portions at 0° C. The resultant was stirred at 0° C. for 30minutes, and then stirred at room temperature for 2 hours. Water (180mL) and 2 N hydrochloric acid (186 mL) were added at 0° C., and theresultant was distillated under reduced pressure. The resulting residuewas extracted with ethyl acetate four times, and the organic layer waswashed with brine and then dried over anhydrous sodium sulfate. Theresultant was distillated under reduced pressure, and the resultingresidue (27.9 g, 90%) was directly used for the subsequent reaction.

Step 2: Benzyl(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane-5-carboxylate

To a solution of the compound obtained in step 1 (27.9 g, 107 mmol) andimidazole (14.5 g, 214 mmol) in dichloromethane (300 mL),tert-butyldimethylsilyl chloride (24.2 g, 160 mmol) was added at roomtemperature, and the resultant was stirred at room temperature for 18hours. The reaction solution was washed with a saturated aqueous citricacid, a saturated aqueous sodium hydrogen carbonate, and brine, driedover anhydrous sodium sulfate, and then distillated under reducedpressure. The resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v) to 50:50 (v/v)] toafford the desired compound (32.5 g, 81%).

¹H-NMR (CDCl₃) δ: 7.39-7.34 (5H, m), 5.23-5.11 (2H, m), 4.10-3.48 (4H,m), 3.16-3.14 (1H, m), 2.15-2.04 (1H, m), 1.81-1.77 (1H, m), 0.91-0.88(9H, m), 0.65-0.55 (4H, m), 0.08-0.01 (6H, m).

MS (APCI) m/z: 376 (M+H)⁺

Step 3:(6S)-6-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]heptane

To a solution of the compound obtained in step 2 (32.5 g, 86.5 mmol) inethanol (400 mL), 7.5% palladium carbon catalyst (moisture content: 54%,5.00 g) was added at room temperature, and the resultant was stirredunder the hydrogen atmosphere at room temperature for 6 hours. Thereaction solution was filtered through a Celite, and the filtrate wasdistillated under reduced pressure to afford the desired compound (21.3g, quantitative).

¹H-NMR (CDCl₃) δ: 3.79-3.77 (1H, m), 3.71-3.69 (1H, m), 3.65-3.60 (1H,m), 3.01-2.98 (2H, m), 1.81-1.71 (2H, m), 0.90 (9H, s), 0.65-0.57 (4H,m), 0.08 (3H, s), 0.07 (3H, s).

MS (APCI, ESI) m/z: 242 (M+H)⁺

Step 4:[(6S)-6-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl](5-methoxy-2-nitro-4-{[tri(propan-2-yl)silyl]oxy}phenyl)methanone

To a solution of 5-methoxy-2-nitro-4-{tri(propan-2-yl)silyl]oxy}benzoicacid (52.2 g, 141 mmol, US 20150283262) and 1-hydroxybenzotriazolemonohydrate (23.8 g, 155 mmol) in dichloromethane (500 mL),N,N′-dicyclohexylcarbodiimide (35.0 g, 170 mmol) was added underice-cooling. The reaction mixture was stirred at room temperature. Afterthe carboxylic acid disappeared, a solution of the compound obtained instep 3 (34.1 g, 141 mmol) and triethylamine (29.4 mL, 212 mmol) indichloromethane (100 mL) was slowly added dropwise thereto. After thereaction solution was stirred at room temperature overnight, saturatedaqueous sodium hydrogen carbonate was added to the reaction mixture, andthe reaction mixture was extracted with chloroform. The organic layerwas washed with water and brine, and dried over anhydrous magnesiumsulfate. The resultant was distillated under reduced pressure, and tothe resulting residue ethyl acetate and diethyl ether were added, andthe solid contents were removed through filtration, and the filtrate wasdistillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [hexane:ethyl acetate=100:0(v/v) to 25:75 (v/v)] to afford the desired compound (55.0 g, 66%).

¹H-NMR (CDCl₃) δ: 7.72-7.66 (1H, m), 6.80-6.73 (1H, m), 4.53-4.49 (1H,m), 4.04-3.95 (1H, m), 3.91-3.88 (3H, m), 3.59-3.54 (1H, m), 3.36-3.25(0.5H, m), 3.01-2.96 (1.5H, m), 2.24-2.20 (0.3H, m), 2.09-2.05 (0.7H,m), 2.00-1.97 (0.7H, m), 1.69-1.67 (0.3H, m), 1.32-1.24 (3H, m),1.12-1.05 (18H, m), 0.93-0.91 (6H, m), 0.79-0.77 (3H, m), 0.71-0.62 (2H,m), 0.57-0.40 (2H, m), 0.12-0.10 (4H, m), 0.11-0.15 (2H, m).

MS (APCI, ESI) m/z: 593 (M+H)⁺

Step 5:(2-Amino-5-methoxy-4-{[tri(propan-2-yl)silyl]oxy}phenyl)[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]methanone

To a solution of the compound obtained in step 4 (55.0 g, 92.8 mmol) inethanol (300 mL), 7.5% palladium carbon (10.0 g) was added under thenitrogen atmosphere. The nitrogen balloon was immediately replaced witha hydrogen balloon, and the reaction mixture was vigorously stirredunder the hydrogen atmosphere at room temperature. After the rawmaterials disappeared, the reaction mixture was filtered, and thefiltrate was distillated under reduced pressure to afford the desiredcompound (52.2 g, 100%), which was directly used for the subsequentreaction.

¹H-NMR (CDCl₃) δ: 6.71 (1H, s), 6.25 (1H, s), 4.55-4.28 (2H, m), 3.97(1H, m), 3.75-3.62 (3H, m), 3.70 (3H, s), 3.09-3.07 (1H, m), 2.24-2.19(1H, m), 1.81-1.68 (1H, m), 1.27-1.22 (3H, m), 1.09-1.05 (18H, m), 0.90(9H, s), 0.65-0.46 (4H, m), 0.07-0.03 (6H, m).

MS (APCI, ESI) m/z: 563 (M+H)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

To a solution of the compound obtained in step 5 (18.6 g, 33.0 mmol) andtriethylamine (6.26 mL, 45.2 mmol) in THF (300 mL), triphosgene (4.22 g,14.2 mmol) was slowly added on an ethanol-ice bath. After the addition,a mixed solution ofN-[(prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide(11.4 g, 30.2 mmol, WO 2011130598) and triethylamine (6.26 mL, 45.2mmol) in THF (100 mL) and N,N-dimethylformamide (30 mL) was slowly addeddropwise to the ice-cooled reaction mixture. After the dropwiseaddition, the ice bath was removed, and the reaction mixture was stirredunder the nitrogen atmosphere at 40° C. After the raw materialsdisappeared, water was added to the reaction mixture, and the reactionmixture was extracted with ethyl acetate. The organic layer was washedwith brine, and dried over anhydrous sodium sulfate. After filtrationfollowed by distillation under reduced pressure, the resulting residuewas purified by silica gel column chromatography [hexane:ethylacetate=100:0 (v/v) to 40:60 (v/v)] to afford the desired compound (23.5g, 74%).

¹H-NMR (CDCl₃) δ: 8.99 (1H, m), 8.58 (1H, s), 7.80 (1H, s), 7.55-7.53(2H, m), 7.34-7.32 (2H, m), 6.77-6.75 (2H, m), 5.94-5.87 (1H, m),5.40-5.38 (1H, m), 5.33-5.29 (1H, m), 5.23-5.21 (1H, m), 5.13 (1H, m),5.10 (2H, m), 4.69-4.64 (1H, m), 4.62-4.52 (2H, m), 4.06-4.03 (1H, m),3.98 (1H, m), 3.76-3.65 (6H, m), 3.04 (1H, m), 2.28-2.26 (1H, m),2.18-2.13 (1H, m), 1.46 (3H, m), 1.32-1.25 (3H, m), 1.11-1.09 (18H, m),0.99-0.84 (15H, m), 0.65-0.40 (4H, m), 0.08-0.00 (6H, m).

MS (APCI, ESI) m/z: 966 (M+H)⁺

Step 7:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

To a solution of the compound obtained in step 6 (23.5 g, 24.3 mmol inTHF (50 mL), methanol (50 mL) and water (44 mL), acetic acid (200 mL)was added at room temperature. The reaction mixture was stirred at roomtemperature. After the raw materials disappeared, the reaction mixturewas extracted with ethyl acetate. The organic layer was washed withwater and brine, and dried over anhydrous sodium sulfate. Afterfiltration followed by distillation under reduced pressure, theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)] to afford the desiredcompound (18.0 g, 87%).

¹H-NMR (CDCl₃) δ: 8.64-8.62 (1H, m), 8.50 (1H, m), 7.69 (1H, m),7.55-7.53 (2H, m), 7.34-7.32 (2H, m), 6.79-6.75 (3H, m), 5.91-5.89 (1H,m), 5.39 (1H, m), 5.32-5.29 (1H, m), 5.23-5.21 (1H, m), 4.68-4.54 (4H,m), 4.31 (1H, m), 4.06-4.04 (1H, m), 3.81-3.79 (3H, m), 3.76 (3H, s),3.63-3.61 (1H, m), 3.13-3.11 (1H, m), 2.16-2.13 (1H, m), 1.87-1.81 (2H,m), 1.46-1.43 (3H, m), 1.30-1.24 (3H, m), 1.12-1.08 (18H, m), 0.98-0.91(6H, m), 0.63-0.45 (4H, m).

MS (APCI, ESI) m/z: 852 (M+H)⁺

Step 8:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of dimethyl sulfoxide (3.75 mL, 52.8 mmol) indichloromethane (300 mL), oxalyl chloride (2.17 mL, 25.3 mmol) wasslowly added dropwise under the nitrogen atmosphere at −78° C. After thedropwise addition, the reaction mixture was stirred at −78° C. Asolution of the compound obtained in step 7 (18.0 g, 21.1 mmol) indichloromethane (50.0 mL) was slowly added to the reaction mixture.Triethylamine (14.6 mL, 105 mmol) was added to the reaction solution at−78° C. After the addition, the refrigerant bath was removed, and thetemperature was slowly raised to room temperature. After the rawmaterials disappeared, water was added to the reaction mixture, and thereaction mixture was extracted with chloroform (200 mL). The organiclayer was washed with water and brine, and dried over anhydrousmagnesium sulfate. After filtration followed by distillation underreduced pressure, the resulting residue was purified by silica gelcolumn chromatography [hexane:ethyl acetate=100:0 (v/v) to 0:60 (v/v)]to afford the desired compound (16.5 g, 92%).

¹H-NMR (CDCl₃) δ: 8.51-8.36 (1H, m), 7.54-7.38 (2H, m), 7.22-7.07 (3H,m), 6.73-6.64 (1H, m), 5.94-5.87 (2H, m), 5.33-5.22 (3H, m), 5.09 (1H,m), 4.97 (1H, m), 4.64-4.58 (4H, m), 4.02-4.00 (1H, m), 3.86-3.83 (3H,m), 3.75-3.70 (1H, m), 3.61-3.54 (2H, m), 3.38-3.29 (1H, m), 2.40 (1H,m), 2.16-2.14 (1H, m), 1.74-1.71 (1H, m), 1.44 (3H, m), 1.18-1.16 (3H,m), 1.05-1.00 (18H, m), 0.97-0.92 (6H, m), 0.72-0.60 (4H, m).

MS (APCI, ESI) m/z: 850 (M+H)⁺

Step 9:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 8 (12.0 g, 14.1 mmol) and2,6-lutidine (6.58 mL, 56.5 mmol) in dichloromethane (200 mL),tert-butyldimethylsilyl trifluoromethylsulfonate (9.73 mL, 42.3 mmol)was slowly added dropwise under the nitrogen atmosphere at 0° C. Afterstirring under ice-cooling for 10 minutes, the ice bath was removed, andstirring was performed at room temperature. After the raw materialsdisappeared, water was added to the reaction mixture, and the reactionmixture was extracted with chloroform. The organic layer was washed withwater and brine, and dried over anhydrous sodium sulfate. Afterfiltration followed by distillation under reduced pressure, theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=100:0 (v/v) to 25:75 (v/v)] to afford the desiredcompound (8.12 g, 60%).

¹H-NMR (CDCl₃) δ: 8.67-8.45 (1H, m), 7.50-7.44 (2H, m), 7.19 (1H, s),7.13 (2H, m), 6.95 (2H, m), 6.62-6.57 (2H, m), 6.01 (1H, m), 5.95-5.86(1H, m), 5.33-5.13 (3H, m), 4.82 (1H, m), 4.65-4.54 (3H, m), 4.03-4.01(1H, m), 3.84-3.82 (3H, m), 3.73-3.66 (1H, m), 3.50-3.48 (1H, m), 3.27(1H, m), 2.37-2.33 (1H, m), 2.19-2.13 (1H, m), 1.54-1.43 (3H, m),1.22-1.13 (3H, m), 1.10-1.00 (18H, m), 0.97-0.91 (6H, m), 0.81 (9H, s),0.76-0.59 (4H, m), 0.19-−0.09 (6H, m).

MS (APCI, ESI) m/z: 964 (M+H)⁺

Step 10:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 9 (8.12 g, 8.42 mmol) inN,N-dimethylformamide (90 mL) and water (2 mL), lithium acetate (0.611g, 9.26 mmol) was added, and the resultant was stirred at roomtemperature. After the raw materials disappeared, water was added to thereaction mixture, which was extracted with ethyl acetate. The organiclayer was washed with water and brine, and dried over anhydrous sodiumsulfate. After filtration followed by distillation under reducedpressure, the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)] toafford the desired compound (5.48 g, 81%).

¹H-NMR (CDCl₃) δ: 8.76-8.60 (1H, m), 8.02-7.56 (1H, m), 7.45-7.44 (2H,m), 7.21 (1H, s), 7.10-7.09 (2H, m), 6.81-6.74 (1H, m), 6.65 (1H, s),6.23 (1H, s), 6.01-5.99 (1H, m), 5.95-5.84 (1H, m), 5.41-5.20 (2H, m),5.16 (1H, m), 4.84 (1H, m), 4.67-4.54 (4H, m), 4.05-4.03 (1H, m), 3.87(3H, s), 3.71 (1H, m), 3.55-3.51 (1H, m), 3.26 (1H, m), 2.35 (1H, m),2.18-2.12 (1H, m), 1.55-1.42 (3H, m), 0.97-0.92 (6H, m), 0.81 (9H, s),0.76-0.61 (4H, m), 0.20-−0.06 (6H, m).

MS (APCI, ESI) m/z: 808 (M+H)⁺

Step 11:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-8′-(3-bromopropoxy)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 10 (2.40 g, 2.97 mmol) was reacted in thesame manner as in step 1 of Example 4 to afford the desired compound(2.73 g, 99%).

¹H-NMR (DMSO-D₆) δ: 10.01-9.86 (1H, m), 8.24-8.04 (2H, m), 7.64-7.54(2H, m), 7.32-7.14 (4H, m), 6.59-6.48 (1H, m), 5.94-5.88 (2H, m),5.32-4.76 (5H, m), 4.44-4.38 (3H, m), 3.87-3.81 (5H, m), 3.64-3.55 (2H,m), 3.41 (1H, m), 3.14 (1H, m), 2.45-2.09 (4H, m), 1.97-1.94 (1H, m),1.44-1.30 (4H, m), 0.89-0.53 (9H, m), 0.79 (9H, s), 0.13-0.06 (6H, m).

MS (APCI, ESI) m/z: 930 [⁸¹Br, (M+H)⁺], 928 [⁷⁹Br, (M+H)⁺].

Example 2: Intermediate 2

Step 1: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycine

To a mixture of glycylglycine (0.328 g, 2.49 mmol),N,N-diisopropylethylamine (0.433 mL, 2.49 mmol) andN,N-dimethylformamide (20 mL),1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidin-2,5-dione (1.00 g, 2.49 mmol,Click Chemistry Tools) and water (10 mL) were added at room temperature,and the resultant was stirred at room temperature overnight. Theresultant was distillated under reduced pressure, and the resultingresidue was purified by silica gel column chromatography[chloroform:CMW=100:0 (v/v) to 0:100 (v/v)] to afford the desiredcompound (0.930 g, 89%). CMW refers to an organic layer for distributionwith chloroform:methanol:water=7:3:1 (v/v/v).

¹H-NMR (DMSO-D₆) δ: 12.58 (1H, s), 8.14-8.12 (1H, m), 8.08-8.07 (1H, m),7.69-7.68 (1H, m), 7.62-7.61 (1H, m), 7.53-7.45 (3H, m), 7.40-7.29 (3H,m), 5.05-5.01 (1H, m), 3.73-3.72 (2H, m), 3.66-3.60 (3H, m), 2.66-2.60(1H, m), 2.33-2.24 (1H, m), 2.08-2.04 (1H, m), 1.81-1.77 (1H, m).

MS (APCI, ESI) m/z: 420[(M+H)⁺].

Step 2: 2,5-Dioxopyrrolidin-1-ylN-[4-(11,12-didehydrodibenzo[b,f]azocin-5 (6H)-yl)-4-oxobutanoyl]glycylglycinate

To a solution of the compound obtained in step 1 (0.612 g, 1.46 mmol))and N-hydroxysuccinimide (0.168 g, 1.459 mmol) in dichloromethane (6mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.420g, 2.19 mmol) was added, and the resultant was stirred at roomtemperature for 21 hours. The resultant was distillated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography [chloroform:CMW=100:0 (v/v) to 0:100 (v/v)] to afford thedesired compound (0.375 g, 50%). CMW refers to an organic layer fordistribution with chloroform:methanol:water=7:3:1 (v/v/v).

Example 3: Drug-Linker 1

Step 1:(2R,11aS)-2-{[tert-Butyl(dimethyl)silyl]oxy}-8-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

To a solution of(2R,11aS)-8-(benzyloxy)-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione(25.5 g, 41.6 mmol, WO 2016149546) in THF (150 mL) and ethanol (150 mL),5% palladium carbon (moisture content: 54%, 10.0 g) was added under thenitrogen atmosphere, and the reaction solution was then stirred underthe hydrogen atmosphere at room temperature for 3 days. Chloroform wasadded to the reaction solution, which was filtered through a Celite, andthe filtrate was then distillated under reduced pressure. The resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=100:0 (v/v) to 50:50 (v/v)] to afford the desired compound (19.4g, 89%).

¹H-NMR (CDCl₃) δ: 7.36 (1H, s), 7.25 (1H, s), 6.01 (1H, s), 5.45-5.43(1H, m), 4.69-4.67 (1H, m), 4.60-4.55 (1H, m), 4.23-4.21 (1H, m), 3.96(3H, s), 3.76-3.68 (2H, m), 3.63-3.61 (1H, m), 3.56-3.53 (1H, m),2.88-2.83 (1H, m), 2.03-2.00 (1H, m), 1.00-0.98 (2H, m), 0.87 (9H, s),0.10 (6H, s), 0.02 (9H, s).

MS (APCI, ESI) m/z: 523 (M+H)⁺

Step 2:(2R,11aS)-8-[(5-Bromopentyl)oxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

To a solution of the compound obtained in step 1 (10.8 g, 20.7 mmol) inN,N-dimethylformamide (30 mL), 1,5-dibromopentane (23.8 g, 103 mmol) andpotassium carbonate (3.43 g, 24.8 mmol) were added at room temperature.After stirring at room temperature for 3 hours, water was added to thereaction solution, which was extracted with ethyl acetate. The organiclayer obtained was washed with brine and dried over sodium sulfate, anddistillated under reduced pressure. The resulting residue was purifiedby silica gel column chromatography [hexane:ethyl acetate=90:10 (v/v) to50:50 (v/v)] to afford the desired compound (14.5 g, quantitative).

¹H-NMR (CDCl₃) δ: 7.34 (1H, s), 7.21 (1H, s), 5.52-5.49 (1H, m),4.63-4.62 (1H, m), 4.58-4.55 (1H, m), 4.24-4.22 (1H, m), 4.07-4.04 (2H,m), 3.92 (3H, s), 3.82-3.64 (3H, m), 3.56-3.53 (1H, m), 3.45-3.43 (2H,m), 2.86-2.84 (1H, m), 2.04-2.00 (1H, m), 1.97-1.87 (4H, m), 1.66-1.62(2H, m), 1.01-0.98 (2H, m), 0.87 (9H, s), 0.10 (6H, s), 0.04 (9H, s).

MS (APCI, ESI) m/z: 673 [⁸¹Br, (M+H)⁺], 671 [⁷⁹Br, (M+H)⁺].

Step 3:(2R,11aS)-8-[(5-Bromopentyl)oxy]-2-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

To a solution of the compound obtained in step 2 (21.5 mmol) in THF (40mL), a 1 mol/L THF solution of tetrabutylammonium fluoride (28.0 mL,28.0 mmol) was added at 0° C. After stirring at room temperature for 30minutes, water was added to the reaction solution, which was extractedwith ethyl acetate, and the organic layer obtained was washed withbrine. The resultant was dried over sodium sulfate, and then distillatedunder reduced pressure. The resulting residue was purified by silica gelcolumn chromatography [chloroform:methanol=97.5:2.5 (v/v) to 92.5:7.5(v/v)] to afford the desired compound (11.3 g, 94%).

¹H-NMR (CDCl₃) δ: 7.34 (1H, s), 7.21 (1H, s), 5.53-5.50 (1H, m),4.69-4.64 (2H, m), 4.32-4.30 (1H, m), 4.10-4.00 (2H, m), 3.91 (3H, s),3.88-3.75 (2H, m), 3.73-3.64 (2H, m), 3.45-3.44 (2H, m), 2.99-2.96 (1H,m), 2.15-2.09 (1H, m), 1.99-1.85 (5H, m), 1.68-1.62 (2H, m), 1.01-0.95(2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 559 [⁸¹Br, (M+H)⁺], 557 [⁷⁹Br, (M+H)⁺].

Step 4:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2,5,11(3H,10H,11aH)-trione

The compound obtained in step 3 (11.3 g, 20.2 mmol), tetrabutylammoniumbromide (0.325 g, 1.01 mmol), and potassium bromide (0.240 g, 2.02 mmol)were dissolved in a saturated aqueous sodium hydrogen carbonate (60mL)/dichloromethane (60 mL), to which nor-AZADO (0.0279 g, 0.202 mmol)and sodium hypochlorite pentahydrate (2.03 g, 27.2 mmol) were added at0° C., and the resultant was stirred at 0° C. for 30 minutes. Becausethe raw materials remained, sodium hypochlorite pentahydrate (1.00 g,13.4 mmol) was added thereto at 0° C., and the resultant was stirred at0° C. for 15 minutes. Sodium hypochlorite pentahydrate (0.300 g, 4.03mmol) was further added thereto at 0° C., and the resultant was stirredat 0° C. for 15 minutes, and the disappearance of the raw materials wasconfirmed by TLC. An aqueous solution of sodium thiosulfate was added tothe reaction solution, which was extracted with chloroform, and theorganic layer obtained was dried over sodium sulfate. The resultant wasdistillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [hexane:ethyl acetate=75:25(v/v) to 40:60 (v/v)] to afford the desired compound (9.74 g, 87%).

¹H-NMR (CDCl₃) δ: 7.33 (1H, s), 7.24 (1H, s), 5.56-5.53 (1H, m),4.71-4.69 (1H, m), 4.66-4.63 (1H, m), 4.27-4.22 (1H, m), 4.12-4.02 (2H,m), 3.93-3.88 (4H, m), 3.82-3.75 (1H, m), 3.69-3.67 (1H, m), 3.61-3.56(1H, m), 3.46-3.44 (2H, m), 2.82-2.77 (1H, m), 1.97-1.89 (4H, m),1.68-1.64 (2H, m), 1.05-0.93 (2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 557 [⁸¹Br, (M+H)⁺], 555 [⁷⁹Br, (M+H)⁺].

Step 5:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yltrifluoromethanesulfonate

To a solution of the compound obtained in step 4 (9.74 g, 17.5 mmol) indichloromethane (160 mL), 2,6-lutidine (8.17 mL, 70.1 mmol) was added at−40° C., and the resultant was stirred at −40° C. for 10 minutes.Anhydrous trifluoromethanesulfonic acid (8.85 mL, 52.6 mmol) was addedto the reaction solution at −40° C., and the resultant was stirred at−40° C. for 30 minutes. To the reaction solution, a 10% aqueous solutionof citric acid was added, which was extracted with chloroform, and theorganic layer obtained was dried over sodium sulfate. The resultant wasdistillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [hexane:ethyl acetate=95:5(v/v) to 70:35 (v/v)] and then purified by NH2 silica gel chromatography[hexane:ethyl acetate=95:5 (v/v) to 65:35 (v/v)] to afford the desiredcompound (7.10 g, 59%).

¹H-NMR (CDCl₃) δ: 7.32 (1H, s), 7.24 (1H, s), 7.15-7.14 (1H, m),5.56-5.53 (1H, m), 4.70-4.68 (1H, m), 4.66-4.63 (1H, m), 4.11-4.01 (2H,m), 3.94-3.90 (4H, m), 3.84-3.75 (1H, m), 3.73-3.68 (1H, m), 3.46-3.44(2H, m), 3.18-3.14 (1H, m), 1.96-1.88 (4H, m), 1.69-1.61 (2H, m),1.02-0.92 (2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 689 [⁸¹Br, (M+H)⁺], 687 [⁷⁹Br, (M+H)⁺].

Step 6:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

To a mixture of the compound obtained in step 5 (2.00 g, 2.91 mmol),4-methoxyphenylboronic acid (0.884 g, 5.82 mmol),tetrakis(triphenylphosphine)palladium (0) (0.336 g, 0.291 mmol) andsodium carbonate (1.23 g, 11.6 mmol), toluene (20 mL), ethanol (10 mL)and water (10 mL) were added at room temperature. The reaction solutionwas stirred at room temperature for 30 minutes, and the reactionsolution was then extracted with ethyl acetate, and the extract waswashed with water and brine. The organic layer was dried over sodiumsulfate, and then distillated under reduced pressure. The resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=90:10 (v/v) to 50:50 (v/v)] to afford the desired compound (1.71g, 91%).

¹H-NMR (CDCl₃) δ: 7.38-7.37 (3H, m), 7.33 (1H, s), 7.25 (1H, s),6.89-6.88 (2H, m), 5.56-5.54 (1H, m), 4.71-4.68 (1H, m), 4.65-4.62 (1H,m), 4.09-4.04 (2H, m), 3.96-3.91 (4H, m), 3.85-3.66 (5H, m), 3.46-3.45(2H, m), 3.16-3.12 (1H, m), 1.99-1.94 (4H, m), 1.69-1.64 (2H, m),1.00-0.98 (2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 647 [⁸¹Br, (M+H)⁺], 645 [⁷⁹Br, (M+H)⁺].

Step 7:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 6 (0.789 g, 1.22 mmol) was dissolved inethanol (10 mL) and THF (10 mL), and 2.0 M tetrahydrofuran solution oflithium borohydride (6.11 mL, 12.2 mmol) was added thereto at 0° C., andthe resultant was stirred at 0° C. for 3 hours. Water was added to thereaction solution, which was extracted with chloroform, and the organiclayer obtained was dried over sodium sulfate. The resultant wasdistillated under reduced pressure, and the resulting residue wasdissolved in dichloromethane (10 mL), ethanol (20 mL) and water (10 mL),to which silica gel (4 g) was added at room temperature, and theresultant was stirred at room temperature for 4 days. The silica gel wasremoved through filtration, and water was added thereto, and theresultant was extracted with chloroform. The organic layer obtained wasdried over sodium sulfate. The resultant was distillated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=60:40 (v/v) to 25:75 (v/v)] toafford the desired compound (0.496 g, 81%).

¹H-NMR (CDCl₃) δ: 7.90-7.89 (1H, m), 7.53 (1H, s), 7.40-7.40 (1H, m),7.35-7.34 (2H, m), 6.92-6.90 (2H, m), 6.83-6.81 (1H, m), 4.43-4.40 (1H,m), 4.13-4.06 (2H, m), 3.96 (3H, s), 3.84 (3H, s), 3.61-3.57 (1H, m),3.47-3.36 (3H, m), 2.00-1.92 (4H, m), 1.67-1.63 (2H, m).

MS (APCI, ESI) m/z: 501 [⁸¹Br, (M+H)⁺], 499 [⁷⁹Br, (M+H)⁺].

Step 8:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

To a solution of the compound obtained in step 7 (0.496 g, 0.992 mmol)in dichloromethane (20 mL), sodium triacetoxyborohydride (0.421 g, 1.99mmol) was added at 0° C. After stirring at room temperature for 2 hours,a saturated aqueous sodium hydrogen carbonate was added thereto, and theresultant was extracted with chloroform. The organic layer was driedover sodium sulfate, and distillated under reduced pressure, and theresulting residue was then purified by silica gel column chromatography[hexane:ethyl acetate=60:40 (v/v) to 25:75 (v/v)] to afford the desiredcompound (0.426 g, 86%).

¹H-NMR (CDCl₃) δ: 7.53-7.53 (2H, m), 7.32-7.30 (2H, m), 6.89-6.87 (2H,m), 6.05 (1H, s), 4.33-4.27 (2H, m), 4.00-3.98 (2H, m), 3.86 (3H, s),3.82 (3H, s), 3.57-3.55 (2H, m), 3.42-3.38 (3H, m), 2.76-2.72 (1H, m),1.96-1.88 (4H, m), 1.65-1.62 (2H, m).

MS (APCI, ESI) m/z: 503 [⁸¹Br, (M+H)⁺], 501 [⁷⁹Br, (M+H)⁺].

Step 9: Prop-2-en-1-yl(11aS)-8-[(5-bromopentyl)oxy]-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

To a solution of the compound obtained in step 8 (0.426 g, 0.849 mmol)in dichloromethane (30 mL), pyridine (0.102 mL 1.27 mmol) and allylchloroformate (0.374 mL, 3.54 mmol) were added at 0° C., and theresultant was stirred at 0° C. for 15 minutes. To the reaction solution,a 10% aqueous solution of citric acid was added, which was extractedwith chloroform, and the organic layer obtained was washed with asaturated aqueous sodium hydrogen carbonate, and then dried over sodiumsulfate. The resultant was distillated under reduced pressure, and theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=90:10 (v/v) to 50:50 (v/v)] to afford the desiredcompound (0.465 g, 94%).

¹H-NMR (CDCl₃) δ: 7.38 (1H, s), 7.31-7.29 (2H, m), 7.26-7.25 (1H, m),6.89-6.87 (2H, m), 6.71 (1H, s), 5.80-5.78 (1H, m), 5.14-5.11 (2H, m),4.65-4.62 (1H, m), 4.39-4.26 (3H, m), 4.03-4.01 (2H, m), 3.92 (3H, s),3.82 (3H, s), 3.66-3.64 (1H, m), 3.46-3.44 (2H, m), 3.30-3.27 (1H, m),2.72-2.68 (1H, m), 1.96-1.88 (4H, m), 1.68-1.60 (2H, m).

MS (APCI, ESI) m/z: 587 [⁸¹Br, (M+H)⁺], 585 [⁷⁹Br, (M+H)⁺].

Step 10:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 10 of Example 1 (0.130 g,0.161 mmol) and the compound obtained in step 9 (0.104 g, 0.177 mmol) inN,N-dimethylformamide (3 mL), potassium carbonate (0.0266 g, 0.193 mmol)was added at room temperature, and the resultant was stirred at roomtemperature overnight. The reaction solution was diluted with ethylacetate, and washed with water and brine, and then dried over sodiumsulfate. The resultant was distillated under reduced pressure, and theresulting residue was then purified by NH2-silica gel columnchromatography [hexane:ethyl acetate=70:30 (v/v) to 0:100 (v/v)] toafford the desired compound (0.184 g, 87%).

¹H-NMR (CDCl₃) δ: 8.76 (1H, s), 7.58-7.56 (2H, m), 7.39 (1H, s),7.32-7.30 (2H, m), 7.26-7.24 (2H, m), 7.19-7.17 (3H, m), 6.90-6.88 (2H,m), 6.78 (1H, s), 6.68-6.66 (1H, m), 6.37 (1H, s), 5.99-5.93 (3H, m),5.34-5.20 (6H, m), 4.66-4.01 (11H, m), 3.90 (3H, s), 3.89 (3H, s),3.78-3.54 (9H, m), 3.31-3.28 (2H, m), 2.73-2.69 (1H, m), 2.38-2.35 (1H,m), 2.19-2.13 (1H, m), 1.82-1.80 (2H, m), 1.46-1.29 (6H, m), 0.98-0.90(6H, m), 0.83 (9H, s), 0.69-0.63 (4H, m), 0.19-0.16 (6H, m).

MS (APCI, ESI) m/z: 1312 (M+H)⁺

Step 11:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 10 (0.1837 g, 0.140 mmol)and acetic acid (0.048 mL, 0.840 mmol) in THF (5.00 mL), a 1 mol/Ltetrahydrofuran solution of tetrabutylammonium fluoride (0.700 mL, 0.700mmol) was added at room temperature, and the resultant was stirred atroom temperature for 3 hours. The reaction solution was diluted withethyl acetate, and the organic layer was washed with a saturated aqueoussodium hydrogen carbonate and brine, and then dried over sodium sulfate.The resultant was distillated under reduced pressure, and the resultingresidue was purified by silica gel chromatography[chloroform:methanol=99.5:0.5 (v/v) to 95:5 (v/v)] to afford the desiredcompound (0.178 g, quantitative).

¹H-NMR (CDCl₃) δ: 8.86 (1H, s), 7.60-7.59 (2H, m), 7.39 (1H, s),7.32-7.20 (7H, m), 6.90-6.88 (2H, m), 6.78 (1H, s), 6.68 (1H, s), 6.38(1H, s), 5.90-5.87 (3H, m), 5.39-5.22 (6H, m), 4.72-4.02 (11H, m), 3.90(3H, s), 3.88 (3H, s), 3.83 (3H, s), 3.70-3.63 (6H, m), 3.32-3.29 (3H,m), 2.73-2.69 (1H, m), 2.43-2.40 (1H, m), 2.12-2.06 (1H, m), 1.77-1.74(2H, m), 1.39-1.25 (6H, m), 0.96-0.89 (6H, m), 0.73-0.66 (4H, m).

MS (APCI, ESI) m/z: 1198 (M+H)⁺

Step 12:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 11 (0.140 mmol) indichloromethane (2 mL), pyrrolidine (0.0579 mL, 0.700 mmol) andtetrakis(triphenylphosphine)palladium (0) (0.0162 g, 0.0140 mmol) wereadded at room temperature, and the resultant was stirred at roomtemperature for 15 minutes. After distillation under reduced pressure,the resulting residue was purified by silica gel chromatography[chloroform:methanol=99.5:0.5 (v/v) to 92.5:7.5 (v/v)] to afford thedesired compound (0.143 g, 99%).

¹H-NMR (CDCl₃) δ: 9.12 (1H, s), 7.94-7.92 (1H, m), 7.57-7.53 (4H, m),7.33-7.31 (2H, m), 7.20-7.18 (3H, m), 6.90-6.88 (2H, m), 6.36 (1H, s),6.07 (1H, s), 5.91-5.88 (1H, m), 5.47-5.44 (1H, m), 5.21-5.13 (1H, m),4.66-4.58 (3H, m), 4.32 (1H, s), 4.03-3.49 (17H, m), 3.38-3.29 (4H, m),3.15-3.14 (1H, m), 2.77-2.73 (1H, m), 2.57 (2H, s), 2.43-2.40 (1H, m),2.32-2.27 (1H, m), 1.81-1.39 (8H, m), 0.98-0.96 (3H, m), 0.85-0.83 (3H,m), 0.75-0.62 (4H, m).

¹H-NMR (CD₃OD, 50° C.) δ: 7.84 (1H, s), 7.56-7.48 (2H, m), 7.44-7.32(4H, m), 7.26-7.13 (3H, m), 6.89 (2H, d, J=8.5 Hz), 6.78-6.66 (1H, m),6.26 (1H, s), 5.96 (1H, d, J=9.7 Hz, H_(11′)), 5.27 (1H, d, J=12.1 Hz),4.96-4.78 (1H, m), 4.63-4.58 (2H, m), 4.49 (1H, q, J=6.9 Hz), 4.28-4.19(1H, m), 4.07-3.89 (4H, m), 3.85 (3H, s), 3.79 (3H, s), 3.76 (3H, s),3.67 (1H, d, J=11.5 Hz), 3.61 (1H, d, J=13.3 Hz), 3.54 (1H, dd, J=9.7,8.2 Hz, H_(11′a)), 3.43-3.31 (2H, m), 3.21 (1H, d, J=11.5 Hz), 3.14 (1H,d, J=4.8 Hz), 2.78 (1H, dd, J=16.6, 4.5 Hz), 2.43 (1H, dd, J=13.0, 8.2Hz, H_(1′b)), 2.05-1.93 (1H, m), 1.91-1.75 (4H, m), 1.73-1.55 (2H, m),1.69 (1H, d, J=13.3 Hz, H_(1′a)), 1.40 (3H, d, J=7.3 Hz), 0.96 (3H, d,J=6.7 Hz), 0.89 (3H, d, J=7.3 Hz), 0.76-0.58 (4H, m).

MS (APCI, ESI) m/z: 1030 (M+H)⁺

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a mixture of the compound obtained in step 1 of Example 2 (0.0640 g,0.153 mmol) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.0446g, 0.180 mmol), dichloromethane (2 mL) was added at room temperature,and the resultant was stirred at room temperature for 15 minutes. To thereaction solution, a solution of the compound obtained in step 13 (0.143g, 0.139 mmol) in dichloromethane (2 mL) was added, and the resultantwas stirred at room temperature for 5 hours, and then distillated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography [chloroform:methanol=99.5:0.5 (v/v) to 92.5:7.5(v/v)] to afford the desired compound (0.103 g, 52%).

Table 1: Peak positions of proton NMR and MS for drug-linker 1

¹H-NMR (DMSO-D₆) δ: 9.93 (1H, s), 8.21-8.16 (2H, m), 8.07-8.04 (1H, m),7.83-7.64 (2H, m), 7.60-7.55 (3H, m), 7.51-7.28 (10H, m), 7.19-7.16 (2H,m), 7.10-7.04 (1H, m), 6.92-6.90 (2H, m), 6.76-6.70 (1H, m), 6.39 (1H,s), 5.77-5.75 (1H, m), 5.21-5.18 (1H, m), 5.03-4.99 (1H, m), 4.82-4.79(1H, m), 4.37-4.35 (1H, m), 4.21-4.20 (2H, m), 4.02-3.24 (26H, m),3.16-3.13 (1H, m), 2.79-2.59 (2H, m), 2.39-2.28 (2H, m), 2.05-1.97 (2H,m), 1.91-1.77 (4H, m), 1.57-1.54 (3H, m), 1.28-1.23 (3H, m), 0.85-0.80(6H, m), 0.67-0.61 (4H, m).

MS (APCI, ESI) m/z: 1431 (M+H)⁺

Example 4: Drug-Linker 2

Step 1:(2R,11aS)-8-(3-Bromopropoxy)-2-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,aH)-dione

The compound obtained in step 1 of Example 3 (5.06 g, 9.67 mmol) and1,3-dibromopropane (4.93 mL, 48.4 mmol) were reacted in the same manneras in step 2 of Example 3 to afford the desired compound (4.85 g, 78%).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.26 (1H, s), 5.52-5.50 (1H, m),4.65-4.63 (1H, m), 4.61-4.55 (1H, m), 4.25-4.14 (3H, m), 3.92 (3H, s),3.82-3.62 (5H, m), 3.57-3.54 (1H, m), 2.86-2.84 (1H, m), 2.41-2.39 (2H,m), 2.06-1.99 (1H, m), 1.03-0.97 (2H, m), 0.87 (9H, s), 0.10 (6H, s),0.04 (9H, s).

MS (APCI, ESI) m/z: 645 [⁸¹Br, (M+H)⁺], 643 [⁷⁹Br, (M+H)⁺].

Step 2:(2R,11aS)-8-(3-Bromopropoxy)-2-hydroxy-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

The compound obtained in step 1 (4.85 g, 7.54 mmol) was reacted in thesame manner as in step 3 of Example 3 to afford the desired compound(4.05 g, quantitative).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.26 (1H, s), 5.53-5.51 (1H, m),4.66-4.61 (2H, m), 4.32-4.30 (1H, m), 4.21-4.16 (2H, m), 3.91-3.85 (4H,m), 3.82-3.74 (1H, m), 3.71-3.59 (4H, m), 2.99-2.96 (1H, m), 2.43-2.37(2H, m), 2.15-2.09 (2H, m), 1.04-0.96 (2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 531 [⁸¹Br, (M+H)⁺], 529 [⁷⁹Br, (M+H)⁺].

Step 3:(11aS)-8-(3-Bromopropoxy)-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2,5,11(3H,10H,11aH)-trione

The compound obtained in step 2 (7.54 mmol) was reacted in the samemanner as in step 4 of Example 3 to afford the desired compound (3.73 g,93%).

¹H-NMR (CDCl₃) δ: 7.34 (1H, s), 7.29 (1H, s), 5.56-5.53 (1H, m),4.72-4.69 (1H, m), 4.67-4.61 (1H, m), 4.23-4.17 (3H, m), 3.97-3.88 (4H,m), 3.82-3.75 (1H, m), 3.74-3.56 (4H, m), 2.82-2.77 (1H, m), 2.43-2.38(2H, m), 1.06-0.94 (2H, m), 0.08-0.00 (9H, m).

Step 4:(11aS)-8-(3-Bromopropoxy)-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yltrifluoromethanesulfonate

The compound obtained in step 3 (3.73 g, 7.08 mmol) was reacted in thesame manner as in step 5 of Example 3 to afford the desired compound(3.27 g, 70%). ¹H-NMR (CDCl₃) δ: 7.33 (1H, s), 7.29 (1H, s), 7.15-7.15(1H, m), 5.56-5.54 (1H, m), 4.70-4.65 (2H, m), 4.21-4.18 (2H, m),3.94-3.91 (4H, m), 3.81-3.79 (1H, m), 3.70-3.64 (3H, m), 3.19-3.15 (1H,m), 2.47-2.38 (2H, m), 1.02-1.00 (2H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 661 [⁸¹Br, (M+H)⁺], 659 [⁷⁹Br, (M+H)⁺].

Step 5:(11aS)-8-(3-Bromopropoxy)-7-methoxy-2-(4-methoxyphenyl-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

The compound obtained in step 4 (3.27 g, 4.96 mmol) was reacted in thesame manner as in step 6 of Example 3 to afford the desired compound(2.49 g, 81%).

¹H-NMR (DMSO-D₆) δ: 7.49-7.47 (2H, m), 7.40 (1H, s), 7.31-7.24 (2H, m),6.93-6.88 (2H, m), 5.33-5.31 (1H, m), 5.25-5.18 (1H, m), 4.81-4.80 (1H,m), 4.23-4.10 (2H, m), 3.85 (3H, s), 3.77 (3H, s), 3.70-3.59 (3H, m),3.52-3.40 (2H, m), 3.15-3.08 (1H, m), 2.33-2.27 (2H, m), 0.86-0.74 (2H,m), −0.07 (9H, s).

MS (APCI, ESI) m/z: 619 [⁸¹Br, (M+H)⁺], 617 [⁷⁹Br, (M+H)⁺].

Step 6:(11aS)-8-(3-Bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 5 (2.49 g, 4.04 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(1.59 g, 84%).

MS (APCI, ESI) m/z: 473 [⁸¹Br, (M+H)⁺], 471 [⁷⁹Br, (M+H)⁺].

Step 7:(11aS)-8-(3-Bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 6 (1.59 g, 3.38 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(1.39 g, 87%).

¹H-NMR (CDCl₃) δ: 7.54 (1H, s), 7.54-7.51 (1H, m), 7.32-7.29 (2H, m),6.89-6.87 (2H, m), 6.10 (1H, s), 4.32-4.28 (2H, m), 4.14-4.13 (2H, m),3.85 (3H, s), 3.82 (3H, s), 3.63-3.62 (2H, m), 3.57-3.55 (2H, m),3.40-3.36 (1H, m), 2.76-2.72 (1H, m), 2.40-2.37 (2H, m).

MS (APCI, ESI) m/z: 475 [⁸¹Br, (M+H)⁺], 473 [⁷⁹Br, (M+H)⁺].

Step 8: Prop-2-en-1-yl(11aS)-8-(3-bromopropoxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][,4]benzodiazepin-10 (5H)-carboxylate

The compound obtained in step 7 (1.40 g, 2.95 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.885 g, 54%).

¹H-NMR (CDCl₃) δ: 7.34 (1H, s), 7.27-7.25 (2H, m), 7.22 (1H, s),6.86-6.84 (2H, m), 6.73 (1H, s), 5.76-5.74 (1H, m), 5.11-5.09 (2H, m),4.62-4.59 (2H, m), 4.33-4.31 (1H, m), 4.16-4.13 (3H, m), 3.88 (3H, s),3.79 (3H, s), 3.60-3.59 (3H, m), 3.27-3.23 (1H, m), 2.69-2.65 (1H, m),2.37-2.34 (2H, m).

MS (APCI, ESI) m/z: 559 [⁸¹Br, (M+H)⁺], 557 [⁷⁹Br, (M+H)⁺].

Step 9:N-{[(Prop-2-en-1-yl)oxy]carbonyl}-L-valyl-N-[4-({[(11′aS)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-{[(prop-2-en-1-yl)oxy]carbonyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11′a-dihydro-1′H,3′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 8 (0.0381 g, 0.0683 mmol) was reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.0712 g, 81%).

MS (APCI, ESI) m/z: 1284 (M+H)⁺.

Step 10:N-{[(Prop-2-en-1-yl)oxy]carbonyl}-L-valyl-N-[4-({[(11′aS)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-{[(prop-2-en-1-yl)oxy]carbonyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11′a-dihydro-1′H,3′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 9 (0.0712 g, 0.0554 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.0671 g, quantitative).

MS (APCI, ESI) m/z: 1170 (M+H)⁺.

Step 11:L-Valyl-N-[4-({[(11′aS)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11′a-dihydro-1′H,3′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 10 (0.0571 mmol) was reacted in the samemanner as in step 12 of Example 3 to afford the desired compound (0.0574g, 99%).

¹H-NMR (CDCl₃) δ: 9.16 (1H, s), 7.93-7.91 (1H, m), 7.55-7.52 (1H, m),7.50-7.47 (3H, m), 7.35-7.32 (2H, m), 7.21 (1H, s), 7.13-7.11 (2H, m),6.90-6.87 (2H, m), 6.40 (1H, s), 6.08 (1H, s), 5.90-5.87 (1H, m),5.37-5.34 (1H, m), 4.73-4.53 (3H, m), 4.23-4.08 (5H, m), 3.89 (3H, s),3.82 (3H, s), 3.78-3.72 (5H, m), 3.57-3.51 (3H, m), 3.38-3.30 (3H, m),2.76-2.71 (1H, m), 2.36-2.24 (4H, m), 1.78-1.42 (6H, m), 1.00-0.98 (3H,m), 0.87-0.84 (3H, m), 0.74-0.62 (4H, m).

MS (APCI, ESI) m/z: 1002 (M+H)⁺.

Step 12: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11′aS)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11′a-dihydro-1′H,3′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 11 (0.189 g, 0.189 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.169 g, 64%).

MS (APCI, ESI) m/z: 1402 (M+H)⁺.

Example 5: Drug-Linker 3

Step 1: 1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-L-isoleucine

To a 1 mol/L aqueous solution of sodium hydroxide (8.80 mL, 8.80 mmol)with a solution of L-propyl-L-isoleucine (1.00 g, 4.40 mmol) in1,4-dioxane (30 mL), allyl chloroformate (0.690 mL, 6.53 mmol) wasslowly added dropwise at 0° C. The reaction mixture was stirred at roomtemperature for 5 hours, and an aqueous solution of potassium hydrogensulfate was then added to the reaction mixture to adjust the pH toaround 4, and the reaction mixture was extracted with chloroform. Theorganic layer was washed with brine, and dried over anhydrous sodiumsulfate. The resultant was filtered and then concentrated under reducedpressure, and hexane was added to the residue. A solid generated wascollected through filtration, and dried to afford the desired compound(1.20 g, 88%).

MS (APCI, ESI) m/z: 311 (M−H)⁻

Step 2:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-[4-(hydroxymethyl)phenyl]-L-isoleucinamide

To THF solution (100 mL) of the compound obtained in step 1 (13.7 g,43.4 mmol) and 4-aminobenzyl alcohol (6.00 g, 48.7 mmol),N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (12.0 g, 48.7 mmol) wasadded at room temperature. The reaction solution was stirred at roomtemperature for 23 hours, to which diethyl ether (200 mL) was added, anda solid generated was then collected through filtration, and thecompound (13.2 g, 65%) obtained was directly used for the subsequentreaction.

Step 3:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-isoleucinamide

The compound obtained in step 2 (6.87 g, 16.5 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(7.46 g, 56%).

¹H-NMR (CDCl₃) δ: 8.99-8.97 (1H, m), 8.45-8.42 (1H, m), 7.81-7.49 (3H,m), 7.36-7.33 (2H, m), 6.81-6.77 (2H, m), 5.96-5.91 (1H, m), 5.32-5.23(2H, m), 5.13-5.10 (2H, m), 4.73-4.30 (6H, m), 4.00-3.98 (1H, m),3.78-3.52 (7H, m), 3.06-3.02 (1H, m), 2.37-2.12 (5H, m), 2.06-1.92 (1H,m), 1.77-1.48 (2H, m), 1.32-1.27 (3H, m), 1.11-1.09 (18H, m), 1.03-0.91(15H, m), 0.66-0.44 (4H, m), 0.09-0.04 (6H, m).

Step 4:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-1{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-isoleucinamide

The compound obtained in step 3 (7.46 g, 7.41 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(6.07 g, 92%).

MS (APCI, ESI) m/z: 892 (M+H)⁺

Step 5:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 4 (6.07 g, 6.80 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(4.18 g, 69%).

MS (APCI, ESI) m/z: 890 (M+H)⁺

Step 6:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 5 (4.18 g, 4.70 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(4.26 g, 90%).

MS (APCI, ESI) m/z: 1004 (M+H)⁺

Step 7:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spirocyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 6 (4.26 g, 4.70 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(2.48 g, 69%).

¹H-NMR (CDCl₃) δ: 8.44-8.41 (1H, m), 7.53-7.37 (1H, m), 7.24-7.23 (1H,m), 7.14-7.11 (2H, m), 6.82 (1H, s), 6.67-6.65 (1H, m), 6.11-6.07 (1H,m), 5.99-5.95 (2H, m), 5.33-5.02 (4H, m), 4.84-4.41 (5H, m), 3.94 (3H,s), 3.73-3.70 (1H, m), 3.59-3.52 (4H, m), 3.29-3.26 (1H, m), 2.39-2.24(5H, m), 1.99-1.97 (2H, m), 1.56-1.53 (1H, m), 1.10-0.64 (19H, m),0.20-0.16 (3H, m), 0.09-0.07 (3H, m).

MS (APCI, ESI) m/z: 848 (M+H)⁺

Step 8:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′-[(trimethylsilyl)oxy]-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 7 (0.200 g, 0.236 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.308 g, 99%).

MS (APCI, ESI) m/z: 1324 (M+H)⁺

Step 9:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 8 (0.308 g, 0.233 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.261 g, 93%).

MS (APCI, ESI) m/z: 1210 (M+H)⁺

Step 10:L-Prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 9 (0.261 g, 0.216 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.183 g, 81%).

¹H-NMR (CDCl₃) δ: 9.06 (1H, s), 8.33-8.31 (1H, m), 7.53-7.47 (4H, m),7.33-7.31 (2H, m), 7.21 (1H, s), 7.11-7.09 (2H, m), 6.89-6.87 (2H, m),6.40 (1H, s), 6.08 (1H, s), 5.91-5.88 (1H, m), 5.35-5.32 (1H, m),4.69-4.66 (2H, m), 4.45-4.28 (3H, m), 4.15-4.05 (3H, m), 3.87 (3H, s),3.82 (3H, s), 3.78 (3H, s), 3.74-3.72 (3H, m), 3.64-3.47 (3H, m),3.37-3.30 (2H, m), 3.04-3.00 (1H, m), 2.94-2.88 (1H, m), 2.75-2.72 (1H,m), 2.42-2.39 (1H, m), 2.13-2.05 (4H, m), 1.92-1.55 (6H, m), 1.20-1.14(1H, m), 0.98-0.96 (3H, m), 0.91-0.89 (3H, m), 0.70-0.66 (4H, m).

MS (APCI, ESI) m/z: 1042 (M+H)⁺

Step 11: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-11H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 10 (0.0474 g, 0.455 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0495 g, 75%).

MS (APCI, ESI) m/z: 1443 (M+H)⁺

Example 6: Drug-Linker 4

Step 1: To a solution of the compound obtained in step 11 of Example 4(0.0564 g, 0.0563 mmol) and triethylamine (0.00936 mL, 0.0675 mmol) inN,N-dimethylformamide (5 mL), 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidin-2,5-dione (0.0249 g, 0.0619 mmol)was added at room temperature. The resultant was stirred at roomtemperature for 2 hours, and then distillated under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [chloroform:methanol=99.5:0.5 (v/v) to 90:10 (v/v)] toafford the desired compound (0.0490 g, 68%).

MS (APCI, ESI) m/z: 1289 (M+H)⁺

Example 7: Drug-Linker 5

Step 1:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-[4-(hydroxymethyl)phenyl]glycinamide

To a solution of starting material 7-1 (1.24 g, 3.80 mmol, Bioorganic &Medicinal Chemistry 2015, 3, 3237-3247) and potassium carbonate (0.945g, 6.84 mmol) in THF (18 mL) and water (12 mL), allyl chloroformate(0.482 mL, 4.560 mmol) was added at 0° C., and added at room temperaturefor 1 hour. After extraction with ethyl acetate, the extract was washedwith water and brine, and dried over sodium sulfate. The resultant wasdistillated under reduced pressure, and the resulting residue was thendissolved in a small amount of ethyl acetate, to which diethyl ether wasadded. A solid generated (1.30 g, 83%) was collected through filtration,and directly used for the subsequent reaction.

Step 2:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]glycinamide

The compound obtained in step 1 (1.30 g, 3.16 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(1.32 g, 54%).

¹H-NMR (CDCl₃) δ: 9.01 (1H, s), 8.38 (1H, s), 7.80 (1H, s), 7.60-7.58(2H, m), 7.32-7.29 (5H, m), 7.19-7.18 (2H, m), 6.76 (1H, s), 6.55 (1H,s), 5.89-5.83 (1H, m), 5.24-5.13 (5H, m), 4.56-4.55 (3H, m), 4.34-4.33(1H, m), 4.10-4.06 (1H, m), 3.98-3.94 (2H, m), 3.75-3.72 (5H, m),3.16-3.08 (3H, m), 2.28-2.25 (1H, m), 1.70-1.68 (1H, m), 1.30-1.27 (3H,m), 1.11-1.09 (18H, m), 0.90 (9H, s), 0.65-0.48 (4H, m), 0.05-0.02 (6H,m).

MS (APCI, ESI) m/z: 1000 (M+H)⁺

Step 3:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]glycinamide

The compound obtained in step 2 (1.32 g, 1.32 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.23 g, quantitative).

¹H-NMR (CDCl₃) δ: 8.48-8.38 (2H, m), 7.71 (1H, s), 7.61-7.59 (2H, m),7.36-7.27 (5H, m), 7.20-7.18 (2H, m), 6.76 (1H, s), 6.55-6.52 (1H, m),5.89-5.83 (1H, m), 5.28-5.13 (5H, m), 4.56-4.55 (3H, m), 4.34-4.33 (1H,m), 4.22-4.20 (1H, m), 4.10-4.06 (1H, m), 3.98-3.94 (1H, m), 3.78-3.75(5H, m), 3.64-3.62 (1H, m), 3.17-3.07 (3H, m), 1.84-1.83 (2H, m),1.30-1.26 (3H, m), 1.11-1.09 (18H, m), 0.61-0.49 (4H, m).

MS (APCI, ESI) m/z: 886 (M+H)⁺

Step 4:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 3 (1.32 mmol) was reacted in the samemanner as in step 8 of Example 1 to afford the desired compound (0.660g, 57%).

¹H-NMR (CDCl₃) δ: 8.34 (1H, s), 7.53-7.51 (2H, m), 7.26-7.18 (8H, m),6.66-6.57 (2H, m), 5.88-5.80 (2H, m), 5.27-5.21 (3H, m), 5.11-5.07 (1H,m), 4.99-4.96 (1H, m), 4.55-4.54 (2H, m), 4.36-4.34 (1H, m), 4.13-3.92(2H, m), 3.83 (3H, s), 3.73-3.70 (1H, m), 3.57-3.55 (1H, m), 3.46-3.44(1H, m), 3.32-3.29 (1H, m), 3.18-3.15 (1H, m), 3.09-3.05 (1H, m),2.42-2.38 (1H, m), 1.75-1.72 (1H, m), 1.25-1.02 (21H, m), 0.73-0.60 (4H,m).

MS (APCI, ESI) m/z: 884 (M+H)⁺

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 4 (0.834 g, 0.943 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.555 g, 59%).

¹H-NMR (CDCl₃) δ: 8.26-8.23 (1H, m), 7.51-7.50 (2H, m), 7.29-7.28 (3H,m), 7.18-7.13 (5H, m), 6.64-6.62 (1H, m), 6.52-6.49 (1H, m), 6.02-6.00(1H, m), 5.88-5.83 (1H, m), 5.25-5.17 (4H, m), 4.84-4.81 (1H, m),4.55-4.55 (2H, m), 4.34-4.33 (1H, m), 4.06-3.97 (2H, m), 3.84 (3H, s),3.71-3.68 (1H, m), 3.50-3.48 (1H, m), 3.28-3.05 (3H, m), 2.36-2.33 (1H,m), 1.56-1.53 (1H, m), 1.28-1.01 (21H, m), 0.81-0.61 (13H, m), 0.19 (3H,s), 0.09 (3H, s).

MS (APCI, ESI) m/z: 998 (M+H)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 5 (0.555 g, 0.556 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.451 g, 96%).

¹H-NMR (CDCl₃) δ: 8.42-8.40 (1H, m), 7.50-7.46 (2H, m), 7.28-7.26 (3H,m), 7.20-7.18 (3H, m), 7.10-7.08 (2H, m), 6.67-6.65 (2H, m), 6.16-6.13(1H, m), 6.02-5.99 (1H, m), 5.88-5.82 (1H, m), 5.28-5.18 (4H, m),4.87-4.84 (1H, m), 4.54-4.53 (2H, m), 4.38-4.36 (1H, m), 4.10-4.07 (1H,m), 3.93-3.90 (4H, m), 3.72-3.69 (1H, m), 3.54-3.52 (1H, m), 3.25-3.17(2H, m), 3.08-3.04 (1H, m), 2.36-2.33 (1H, m), 1.57-1.54 (1H, m),0.81-0.61 (13H, m), 0.19 (3H, s), 0.10 (3H, s).

MS (APCI, ESI) m/z: 842 (M+H)⁺

Step 7:N-[(Prop-2-en-1-yloxy)carbonyl]-L-phenylalanyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 6 (0.115 g, 0.137 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.160 g, 89%)

MS (APCI, ESI) m/z: 1318 (M+H)⁺

Step 8:N-[(Prop-2-en-1-yl)carbonyl]-L-phenylalanyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 7 (0.160 g, 0.121 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.136 g, 93%).

MS (APCI, ESI) m/z: 1204 (M+H)⁺

Step 9:L-Phenylalanyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 8 (0.136 g, 0.113 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.0372 g, 32%).

¹H-NMR (CDCl₃) δ: 8.84-8.81 (1H, m), 8.07-8.05 (1H, m), 7.53-7.39 (4H,m), 7.34-7.19 (8H, m), 7.12-7.10 (2H, m), 6.90-6.87 (2H, m), 6.44-6.42(1H, m), 6.10-6.08 (1H, m), 5.90-5.88 (1H, m), 5.38-5.35 (1H, m),4.76-4.72 (1H, m), 4.57-4.44 (1H, m), 4.32-4.29 (1H, m), 4.17-4.01 (5H,m), 3.89-3.52 (17H, m), 3.41-3.25 (3H, m), 2.72-2.69 (2H, m), 2.43-2.40(1H, m), 2.19-2.16 (2H, m), 1.78-1.74 (1H, m), 1.59-1.56 (1H, m),0.72-0.66 (4H, m).

MS (APCI, ESI) m/z: 1036 (M+H)⁺

Step 10: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}glycinamide

The compound obtained in step 9 (0.0372 g, 0.0359 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0170 g, 33%).

MS (APCI, ESI) m/z: 1437 (M+H)⁺

Example 8: Drug-Linker 6

Step 1:N-[(11,12-Didehydro-5,6-dihydrodibenzo[a,e][8]annulen-5-yloxy)carbonyl]glycylglycine

To a solution of 11,12-didehydro-5,6-dihydrodibenzo[a,e][8]annulen-5-yl4-nitrophenylcarbamate (0.437 g, 1.14 mmol) andN,N-diisopropylethylamine (0.198 mL, 1.14 mmol) in N,N-dimethylformamide(6 mL), glycylglycine (0.150 g, 1.14 mmol) and water (3 mL) were addedat room temperature, and the resultant was stirred at room temperatureovernight. The resultant was distillated under reduced pressure, and theresulting residue was purified by silica gel column chromatography[chloroform:CMW=100:0 (v/v) to 0:100 (v/v)] to afford the desiredcompound (0.324 g, 75%).

MS (APCI, ESI) m/z: 378 (M+H)⁺

Step 2:N-[(11,12-Didehydro-5,6-dihydrodibenzo[a,e][8]annulen-5-yloxy)carbonyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(1aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 1 (0.0306 g, 0.0305 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0361 g, 87%).

MS (APCI, ESI) m/z: 1362 (M+H)⁺

Example 9: Drug-Linker 7

Step 1:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-([{(2-{[((6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2,4]hept-5-yl]carbonyl)-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-N⁵-carbamoyl-L-ornithinamide

Starting material 9-1 was reacted in the same manner as in step 6 ofExample 1 to afford the desired compound (1.37 g, 45%).

¹H-NMR (DMSO-D₆) δ: 10.06 (1H, s), 9.16 (1H, s), 8.10-8.06 (1H, m),7.62-7.60 (2H, m), 7.33-7.31 (2H, m), 7.27-7.24 (2H, m), 6.84-6.81 (1H,m), 5.94-5.89 (2H, m), 5.41 (2H, s), 5.32-5.28 (1H, m), 5.18-5.16 (1H,m), 5.03 (2H, s), 4.48-4.42 (3H, m), 4.30 (1H, s), 3.93-3.73 (6H, m),3.47-3.14 (3H, m), 3.00-2.95 (2H, m), 2.00-1.89 (2H, m), 1.65-1.60 (2H,m), 1.42-1.39 (2H, m), 1.26-1.19 (3H, m), 1.04-1.01 (18H, m), 0.88-0.75(15H, m), 0.51-0.49 (4H, m), 0.05-0.17 (6H, m).

MS (APCI, ESI) m/z: 1052 (M+H)⁺

Step 2:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide

The compound obtained in step 1 (1.37 g, 1.31 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.00 g, 82%).

¹H-NMR (DMSO-D₆) δ: 10.07 (1H, s), 9.13 (1H, s), 8.11-8.09 (1H, m),7.62-7.60 (2H, m), 7.34-7.31 (2H, m), 7.26-7.23 (2H, m), 6.92-6.90 (1H,m), 5.97-5.86 (2H, m), 5.41 (2H, s), 5.32-5.28 (1H, m), 5.19-5.16 (1H,m), 5.04 (2H, s), 4.80 (1H, s), 4.48-4.41 (3H, m), 4.27 (1H, s),3.93-3.87 (1H, m), 3.74 (3H, s), 3.61-3.58 (2H, m), 3.39-3.30 (2H, m),3.03-2.97 (3H, m), 2.00-1.84 (2H, m), 1.65-1.60 (2H, m), 1.44-1.37 (2H,m), 1.26-1.19 (3H, m), 1.05-1.04 (18H, m), 0.87-0.83 (6H, m), 0.53-0.42(4H, m).

MS (APCI, ESI) m/z: 938 (M+H)⁺

Step 3:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxy-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide

To a mixture of the compound obtained in step 2 (1.00 g, 1.07 mmol),dichloromethane (80 mL), and dimethylformamide (10 mL), Dess-Martinperiodinane (0.455 g, 1.07 mmol) was added at 0° C. After stirring at 0°C. for 1 hour, Dess-Martin periodinane (0.455 g, 1.07 mmol) was againadded thereto, and the resultant was stirred at 0° C. for 1 hour. To thereaction solution, a saturated aqueous sodium hydrogen carbonate wasadded, and the resultant was extracted with chloroform, and the extractwas then washed with brine. The resultant was distillated under reducedpressure, to which ethyl acetate was then added, and a solid wascollected through filtration. The filtrate was distillated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=50:50 to hexane:ethyl acetate=0:100(v/v)] and combined with the solid to afford the desired compound (0.671g, 67%).

¹H-NMR (DMSO-D₆) δ: 10.05 (1H, s), 8.11-8.09 (1H, m), 7.56-7.54 (2H, m),7.25-7.23 (1H, m), 7.13-7.09 (3H, m), 6.62 (1H, s), 6.53 (1H, s),5.94-5.88 (2H, m), 5.78-5.76 (1H, m), 5.40 (2H, s), 5.32-5.28 (1H, m),5.17-5.14 (2H, m), 4.81-4.78 (1H, m), 4.48-4.41 (3H, m), 3.92-3.90 (1H,m), 3.79 (3H, s), 3.54-3.51 (1H, m), 3.16-3.14 (1H, m), 3.02-2.89 (2H,m), 2.37-2.34 (2H, m), 1.97-1.92 (1H, m), 1.63-1.57 (3H, m), 1.43-1.37(2H, m), 1.08-1.01 (21H, m), 0.87-0.83 (6H, m), 0.67-0.61 (4H, m).

MS (APCI, ESI) m/z: 938 (M+H)⁺

Step 4:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

Using a mixed solvent of dichloromethane (80 mL) and dimethylformamide(5 mL), the compound obtained in step 3 (0.671 g, 0.712 mmol) wasreacted in the same manner as in step 9 of Example 1 to afford thedesired compound (0.335 g, 44%).

¹H-NMR (DMSO-D₆) δ: 10.04 (1H, s), 8.12-8.10 (1H, m), 7.56-7.54 (2H, m),7.26-7.24 (1H, m), 7.14-7.11 (3H, m), 6.51 (1H, s), 5.94-5.91 (3H, m),5.40-5.16 (5H, m), 4.79-4.76 (1H, m), 4.47-4.44 (3H, m), 3.92-3.90 (1H,m), 3.80 (3H, s), 3.55-3.52 (1H, m), 3.17-3.14 (1H, m), 3.00-2.96 (3H,m), 2.56-2.30 (1H, m), 2.06-1.17 (6H, m), 1.10-0.99 (21H, m), 0.78-0.61(19H, m), 0.17 (3H, s), 0.07 (3H, s).

MS (APCI, ESI) m/z: 1050 (M+H)⁺

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

The compound obtained in step 4 (0.355 g, 0.712 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.264 g, 93%).

¹H-NMR (DMSO-D₆) δ: 10.07-10.03 (1H, m), 9.89-9.86 (1H, m), 8.12-8.10(1H, m), 7.63-7.54 (2H, m), 7.35-7.26 (1H, m), 7.14-7.12 (2H, m), 7.06(1H, s), 6.62-6.59 (1H, m), 5.97-5.87 (3H, m), 5.43-5.40 (2H, m),5.32-5.28 (1H, m), 5.17-5.14 (2H, m), 4.86-4.82 (1H, m), 4.46-4.42 (3H,m), 3.91-3.89 (1H, m), 3.81 (3H, s), 3.54-3.51 (1H, m), 3.42-3.40 (1H,m), 3.09-2.96 (3H, m), 2.40-2.34 (1H, m), 1.98-1.97 (1H, m), 1.68-1.59(2H, m), 1.42-1.38 (3H, m), 0.77-0.64 (19H, m), 0.16 (3H, s), 0.08 (3H,s).

MS (APCI, ESI) m/z: 894 (M+H)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁵-carbamoyl-L-ornithinamide

The compound obtained in step 5 (0.113 g, 0.126 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.149 g, 86%).

MS (APCI, ESI) m/z: 1370 (M+H)⁺

Step 7:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N⁵-carbamoyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide

The compound obtained in step 6 (0.149 g, 0.109 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.119 g, 87%).

MS (APCI, ESI) m/z: 1256 (M+H)⁺

Step 8:L-Valyl-N⁵-carbamoyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide

The compound obtained in step 7 (0.050 g, 0.0398 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0347 g, 80%).

¹H-NMR (CDCl₃) δ: 9.43 (1H, s), 7.96-7.94 (1H, m), 7.51-7.46 (4H, m),7.33-7.31 (2H, m), 7.22 (1H, s), 7.13-7.11 (2H, m), 6.90-6.87 (2H, m),6.46 (1H, s), 6.11 (1H, s), 5.92-5.89 (1H, m), 5.42-5.39 (2H, m),4.78-4.67 (4H, m), 4.31-4.29 (1H, m), 4.11-4.04 (3H, m), 3.92-3.70 (13H,m), 3.60-3.23 (8H, m), 3.07-3.05 (1H, m), 2.75-2.70 (1H, m), 2.43-2.39(1H, m), 2.19-2.16 (3H, m), 1.73-1.48 (6H, m), 0.98-0.96 (3H, m),0.83-0.81 (3H, m), 0.71-0.65 (4H, m).

MS (APCI, ESI) m/z: 1088 (M+H)⁺

Step 9: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N⁵-carbamoyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide

The compound obtained in step 8 (0.0347 g, 0.0319 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.00650 g, 14%).

MS (APCI, ESI) m/z: 1489 (M+H)⁺

Example 10: Drug-Linker 8

Step 1:N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-valyl-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysine

To a solution of starting material 10-1 (2.78 g, 7.75 mmol, Bioscience,Biotechnology, and Biochemistry 2012, 76, 205) in 1,2-dimethoxyethane(30 mL), water (30 mL), and THF (15 mL), sodium hydrogen carbonate (1.30g, 15.5 mmol) and 2,5-dioxopyrrolidin-1-ylN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valinate (3.39 g, 7.76 mmol) wereadded at room temperature. The reaction solution was stirred at roomtemperature for five days, and then extracted with a mixed liquid ofchloroform and methanol (10:1, v/v). The organic layer was washed withwater and brine, and then distillated under reduced pressure. Theresulting residue was washed with diethyl ether, and a solid was removedthrough filtration. The filtrate was distillated under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=30:70 (v/v) to 0:100 (v/v)] toafford the desired compound (2.13 g, 43%).

¹H-NMR (CDCl₃) δ: 7.78-7.76 (2H, m), 7.60-7.58 (2H, m), 7.41-7.39 (2H,m), 7.32-7.30 (2H, m), 6.85-6.83 (1H, m), 5.58-5.56 (1H, m), 5.32-5.30(1H, m), 4.72-4.57 (3H, m), 4.46-4.34 (2H, m), 4.23-4.21 (1H, m),4.05-4.03 (1H, m), 3.22-3.15 (2H, m), 2.06-1.88 (3H, m), 1.52-1.51 (2H,m), 1.40-1.38 (2H, m), 0.97-0.96 (6H, m).

MS (APCI, ESI) m/z: 642 (M+H)⁺

Step 2:N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 1 (2.11 g, 3.29 mmol) was reacted in thesame manner as in step 2 of Example 5, and the resulting compound (2.24g, 91%) was directly used for the subsequent reaction.

Step 3:L-Valyl-N-[4-(hydroxymethyl)phenyl]-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

To a solution of the compound obtained in step 2 (2.24 g, 3.00 mmol) inN,N-dimethylformamide (20 mL), piperidine (0.5934 mL, 5.994 mmol) wasadded at room temperature, and the resultant was stirred at roomtemperature for 1 hour. The resultant was distillated under reducedpressure, and the resulting residue (1.576 g, quantitative) was directlyused for the subsequent reaction.

Step 4:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-(hydroxymethyl)phenyl]-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 3 (1.58 g, 3.00 mmol) was reacted in thesame manner as in step 1 of Example 7, and the resulting compound (1.50g, 82%) was directly used for the subsequent reaction.

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 4 (1.57 g, 2.57 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(1.691 g, 71%).

¹H-NMR (CDCl₃) δ: 9.04-9.02 (1H, m), 8.48-8.45 (1H, m), 7.81 (1H, s),7.55-7.53 (2H, m), 7.35-7.33 (2H, m), 6.76 (1H, s), 6.68-6.66 (1H, m),5.94-5.86 (1H, m), 5.32-5.23 (4H, m), 5.14-5.10 (2H, m), 4.79-4.76 (1H,m), 4.69-4.67 (1H, m), 4.57-4.54 (4H, m), 4.03-4.02 (2H, m), 3.75-3.72(5H, m), 3.29-3.22 (2H, m), 3.04-3.02 (1H, m), 2.27-2.01 (4H, m),1.83-1.58 (3H, m), 1.46-1.44 (2H, m), 1.31-1.27 (3H, m), 1.11-1.09 (18H,m), 1.00-0.90 (15H, m), 0.65-0.48 (4H, m), 0.06-0.03 (6H, m).

MS (APCI, ESI) m/z: 1219 (M+Na)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 5 (1.69 g, 1.41 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.43 g, 94%).

¹H-NMR (CDCl₃) δ: 8.57-8.52 (2H, m), 7.70 (1H, s), 7.56-7.54 (2H, m),7.35-7.33 (2H, m), 6.76-6.75 (2H, m), 5.91-5.90 (1H, m), 5.40-5.26 (4H,m), 5.12 (2H, s), 4.78-4.75 (1H, m), 4.69-4.66 (1H, m), 4.58-4.55 (4H,m), 4.37-4.34 (1H, m), 4.04-4.02 (1H, m), 3.80-3.77 (5H, m), 3.65-3.62(1H, m), 3.28-3.11 (3H, m), 2.13-2.04 (2H, m), 1.81-1.78 (3H, m),1.60-1.58 (2H, m), 1.45-1.43 (2H, m), 1.33-1.25 (3H, m), 1.11-1.09 (18H,m), 0.98-0.94 (6H, m), 0.58-0.51 (4H, m).

MS (APCI, ESI) m/z: 1083 (M+H)⁺

Step 7:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 6 (1.43 g, 1.32 mmol) was reacted in thesame manner as in step 3 of Example 9 to afford the desired compound(0.714 g, 50%).

¹H-NMR (CDCl₃) δ: 8.49-8.46 (1H, m), 7.52-7.45 (2H, m), 7.19-7.18 (3H,m), 6.72-6.68 (2H, m), 5.90-5.87 (2H, m), 5.33-5.23 (4H, m), 5.10-5.07(1H, m), 4.98-4.95 (1H, m), 4.78-4.76 (1H, m), 4.69-4.66 (1H, m),4.58-4.53 (3H, m), 4.01 (1H, s), 3.83-3.81 (4H, m), 3.73-3.70 (1H, m),3.57-3.55 (2H, m), 3.29-3.25 (3H, m), 2.42-2.39 (1H, m), 2.15-2.13 (1H,m), 2.03-2.01 (2H, m), 1.74-1.71 (2H, m), 1.44-1.42 (2H, m), 1.23-1.17(3H, m), 1.03-0.93 (24H, m), 0.67-0.64 (4H, m).

MS (APCI, ESI) m/z: 1081 (M+H)⁺

Step 8:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N⁶-[tert-butyl(dimethyl)silyl]-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 7 (0.714 g, 0.660 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.476 g, 60%).

¹H-NMR (CDCl₃) δ: 8.63-8.51 (1H, m), 7.49-7.48 (2H, m), 7.18-7.14 (3H,m), 6.61-6.53 (2H, m), 5.99-5.94 (2H, m), 5.33-5.17 (4H, m), 4.81-4.78(3H, m), 4.59-4.57 (3H, m), 4.03-4.01 (1H, m), 3.88-3.85 (4H, m),3.70-3.67 (2H, m), 3.50-3.47 (1H, m), 3.24-3.17 (3H, m), 2.37-2.34 (1H,m), 2.13-2.07 (2H, m), 1.59-1.54 (3H, m), 1.38 (2H, s), 1.16-0.92 (35H,m), 0.81-0.76 (9H, m), 0.67-0.64 (4H, m), 0.34-0.31 (6H, m), 0.19 (3H,s), 0.09 (3H, s).

Step 9:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N⁶-[tert-butyl(dimethyl)silyl]-N-{4-[({[(11′S,11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 8 (0.476 g, 0.398 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.283 g, 68%).

¹H-NMR (CDCl₃) δ: 8.48 (1H, s), 7.51-7.47 (2H, m), 7.25-7.24 (2H, m),7.12-7.10 (2H, m), 6.70-6.67 (2H, m), 6.09-6.07 (1H, m), 5.98-5.92 (2H,m), 5.33-5.20 (5H, m), 4.82-4.71 (3H, m), 4.59-4.56 (3H, m), 4.03-4.00(1H, m), 3.91 (3H, s), 3.72-3.69 (1H, m), 3.54-3.52 (1H, m), 3.28-3.25(3H, m), 2.37-2.34 (1H, m), 2.18-2.16 (1H, m), 2.05-1.99 (1H, m),1.78-1.75 (1H, m), 1.56-1.53 (2H, m), 1.43-1.41 (2H, m), 0.98-0.94 (6H,m), 0.82-0.75 (9H, m), 0.67-0.64 (4H, m), 0.19 (3H, s), 0.10 (3H, s).

MS (APCI, ESI) m/z: 1039 (M+H)⁺

Step 10:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 9 (0.119 g, 0.114 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.134 g, 77%).

Step 11:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 10 (0.134 g, 0.0881 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.120 g, 97%).

MS (APCI, ESI) m/z: 1423 (M+Na)⁺

Step 12:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 11 (0.120 g, 0.0855 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0813 g, 77%).

¹H-NMR (CDCl₃) δ: 9.10 (1H, s), 7.94-7.92 (1H, m), 7.58 (1H, s),7.47-7.45 (3H, m), 7.35-7.33 (2H, m), 7.21 (2H, s), 7.13-7.11 (2H, m),6.90-6.88 (2H, m), 6.43 (1H, s), 6.11 (1H, s), 5.90-5.88 (1H, m), 5.51(1H, s), 5.39-5.36 (1H, m), 4.73-4.70 (3H, m), 4.52-4.51 (2H, m), 4.32(1H, s), 4.13-4.08 (3H, m), 3.89 (3H, s), 3.80-3.76 (9H, m), 3.60-3.50(4H, m), 3.34-3.24 (5H, m), 2.76-2.72 (1H, m), 2.44-2.12 (4H, m),1.94-1.27 (7H, m), 1.00-0.98 (3H, m), 0.84-0.82 (3H, m), 0.70-0.66 (4H,m).

MS (APCI, ESI) m/z: 1233 (M+H)⁺

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-N′-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinamide

The compound obtained in step 12 (0.0813 g, 0.0659 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0721 g, 67%).

MS (APCI, ESI) m/z: 1656 (M+Na)⁺

Step 14: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-lysinamide

The compound obtained in step 13 (0.0721 g, 0.0441 mmol) was reacted inthe same manner as in step 6 of Example 21 to afford the desiredcompound (0.0348 g, 54%).

MS (APCI, ESI) m/z: 1460 (M+H)⁺

Example 11: Drug-Linker 9

Step 1: N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-D-valyl-L-alanine

L-alanine (0.0721 g, 0.0441 mmol) was reacted in the same manner as instep 1 of Example 10, except that 2,5-dioxopyrrolidin-1-ylN-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-valinate (0.528 g, 5.92 mmol) wasused in place of 2,5-dioxopyrrolidin-1-ylN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valinate. The resulting compound(0.0348 g, 54%) was directly used for the subsequent reaction.

¹H-NMR (DMSO-D₆) δ: 10.55 (1H, s), 8.23-8.21 (1H, m), 7.91-7.89 (2H, m),7.76-7.75 (2H, m), 7.42-7.40 (3H, m), 7.33-7.31 (2H, m), 4.31-4.20 (4H,m), 3.93-3.91 (1H, m), 1.97-1.93 (1H, m), 1.27-1.25 (3H, m), 0.86-0.84(6H, m).

MS (APCI, ESI) m/z: 411 (M+H)⁺

Step 2:N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-D-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide

The compound obtained in step 1 (2.05 g, 4.99 mmol) was reacted in thesame manner as in step 2 of Example 5, and the resulting compound (2.19g, 85%) was directly used for the subsequent reaction.

Step 3: D-Valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide

The compound obtained in step 2 (2.19 g, 4.25 mmol) was reacted in thesame manner as in step 3 of Example 10, and the resulting compound(0.966 g, 76%) was directly used for the subsequent reaction.

Step 4:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-[4-(hydroxymethyl)phenyl]-L-alaninamide

The compound (0.966 g, 3.29 mmol) obtained in step 3 was reacted in thesame manner as in step 1 of Example 7, and the resulting compound (1.11g, 89%) was directly used for the subsequent reaction.

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 4 (1.11 g, 2.93 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(1.75 g, 80%).

¹H-NMR (CDCl₃) δ: 9.02 (1H, s), 8.55 (1H, s), 7.81 (1H, s), 7.57-7.54(2H, m), 7.34-7.32 (2H, m), 6.76 (1H, s), 6.52-6.50 (1H, m), 5.91-5.86(1H, m), 5.30-5.22 (3H, m), 5.13-5.10 (2H, m), 4.65-4.59 (4H, m),3.99-3.97 (1H, m), 3.87-3.85 (1H, m), 3.75-3.72 (5H, m), 3.04-3.02 (1H,m), 2.28-2.13 (2H, m), 1.70-1.68 (1H, m), 1.49-1.47 (3H, m), 1.31-1.27(3H, m), 1.11-1.09 (18H, m), 1.00-0.90 (15H, m), 0.65-0.48 (4H, m),0.06-0.03 (6H, m).

MS (APCI, ESI) m/z: 966 (M+H)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 5 (1.75 g, 1.81 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.53 g, 99%).

¹H-NMR (CDCl₃) δ: 8.66 (1H, s), 8.50 (1H, s), 7.69 (1H, s), 7.57-7.54(2H, m), 7.34-7.32 (2H, m), 6.75-6.71 (2H, m), 5.90-5.85 (1H, m),5.40-5.38 (1H, m), 5.29-5.21 (2H, m), 5.12 (2H, s), 4.71-4.50 (4H, m),4.34-4.31 (1H, m), 3.89-3.77 (6H, m), 3.64-3.61 (1H, m), 3.13-3.10 (1H,m), 2.17-2.09 (1H, m), 1.87-1.84 (2H, m), 1.48-1.46 (3H, m), 1.32-1.28(3H, m), 1.11-1.09 (18H, m), 0.97-0.94 (6H, m), 0.63-0.49 (4H, m).

MS (APCI, ESI) m/z: 852 (M+H)⁺

Step 7:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-{4-[({[(11′S,11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 6 (1.53 g, 1.79 mmol) was reacted in thesame manner as in step 3 of Example 9 to afford the desired compound(1.24 g, 81%).

¹H-NMR (CDCl₃) δ: 8.51 (1H, s), 7.51-7.49 (2H, m), 7.18-7.15 (3H, m),6.65 (1H, s), 6.56-6.54 (1H, m), 5.90-5.85 (2H, m), 5.31-5.19 (3H, m),5.10-5.07 (1H, m), 4.97-4.94 (1H, m), 4.67-4.50 (3H, m), 3.90-3.88 (1H,m), 3.84 (3H, s), 3.73-3.70 (1H, m), 3.58-3.56 (2H, m), 3.31-3.28 (1H,m), 2.42-2.39 (1H, m), 2.18-2.15 (1H, m), 1.74-1.71 (1H, m), 1.48-1.46(3H, m), 1.19-0.88 (27H, m), 0.69-0.65 (4H, m).

MS (APCI, ESI) m/z: 850 (M+H)⁺

Step 8:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (1.24 g, 1.45 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.979 g, 70%).

¹H-NMR (CDCl₃) δ: 8.48 (1H, s), 7.51-7.49 (2H, m), 7.19 (1H, s),7.14-7.12 (2H, m), 6.62 (1H, s), 6.53-6.51 (1H, m), 6.01-5.99 (1H, m),5.91-5.85 (1H, m), 5.30-5.28 (2H, m), 5.21-5.15 (2H, m), 4.82-4.79 (1H,m), 4.68-4.51 (3H, m), 3.88-3.84 (4H, m), 3.71-3.69 (1H, m), 3.50-3.47(1H, m), 3.28-3.25 (1H, m), 2.37-2.34 (1H, m), 2.20-2.13 (1H, m),1.52-1.47 (4H, m), 1.21-0.94 (27H, m), 0.80-0.77 (9H, m), 0.67-0.64 (4H,m), 0.19 (3H, s), 0.08 (3H, s).

MS (APCI, ESI) m/z: 964 (M+H)⁺

Step 9:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 8 (0.979 g, 1.02 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.769 g, 94%).

¹H-NMR (CDCl₃) δ: 8.71 (1H, s), 7.37-7.35 (2H, m), 7.23 (1H, s),7.04-7.02 (2H, m), 6.86-6.84 (1H, m), 6.74-6.72 (1H, m), 6.65 (1H, s),6.05-5.85 (2H, m), 5.64-5.62 (1H, m), 5.32-5.20 (3H, m), 4.82-4.78 (1H,m), 4.70-4.52 (3H, m), 4.00-3.98 (1H, m), 3.93-3.90 (3H, m), 3.73-3.70(1H, m), 3.55-3.53 (1H, m), 3.27-3.23 (1H, m), 2.38-2.18 (2H, m),1.60-1.46 (4H, m), 1.00-0.92 (6H, m), 0.80 (9H, s), 0.68-0.63 (4H, m),0.20 (3H, s), 0.10 (3H, s).

MS (APCI, ESI) m/z: 808 (M+H)⁺

Step 10:N-[(Prop-2-ene)-1-yloxy)carbonyl]-D-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 9 (0.100 g, 0.124 mmol) was used reactedin the same manner as in step 9 of Example 4 to afford the desiredcompound (0.148 g, 94%).

MS (APCI, ESI) m/z: 1284 (M+H)⁺

Step 11:N-[(Prop-2-en-1-yloxy)carbonyl]-D-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 10 (0.148 g, 0.124 mmol) was used andreacted in the same manner as in step 11 of Example 3 to afford thedesired compound (0.132 g, 98%).

MS (APCI, ESI) m/z: 1170 (M+H)⁺

Step 12:D-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 11 (0.132 g, 0.113 mmol) was used andreacted in the same manner as in step 12 of Example 3 to afford thedesired compound (0.0963 g, 85%)

¹H-NMR (CDCl₃) δ: 9.12 (1H, s), 7.85-7.84 (1H, m), 7.54-7.52 (1H, m),7.49 (1H, s), 7.44-7.42 (2H, m), 7.34-7.32 (2H, m), 7.21 (1H, s),7.13-7.11 (2H, m), 6.90-6.88 (2H, m), 6.41 (1H, s), 6.10 (1H, s),5.90-5.87 (1H, m), 5.35-5.32 (1H, m), 4.74-4.71 (1H, m), 4.60-4.56 (2H,m), 4.30 (1H, s), 4.13-4.10 (4H, m), 3.89 (3H, s), 3.83 (3H, s), 3.80(3H, s), 3.74-3.71 (1H, m), 3.60-3.49 (4H, m), 3.39-3.35 (1H, m),3.31-3.27 (2H, m), 2.75-2.72 (1H, m), 2.44-2.18 (4H, m), 1.78-1.44 (6H,m), 0.98-0.97 (3H, m), 0.74-0.68 (7H, m).

MS (APCI, ESI) m/z: 1002 (M+H)⁺

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-D-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 12 (0.0455 g, 0.0454 mmol) was used andreacted in the same manner as in step 13 of Example 3 to afford thedesired compound (0.0416 g, 65%).

MS (APCI, ESI) m/z: 1403 (M+H)⁺

Example 12: Drug-Linker 10

Step 1: Compound 12-2

To starting material 12-1 (2.01 g, 5.94 mmol) in dichloromethane (50mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.37g, 7.13 mmol) was added at room temperature, and the resultant wasstirred at room temperature for 10 minutes, and N-hydroxysuccinimide(0.821 g, 7.13 mmol) was then added thereto at 0° C. The reactionsolution was stirred at room temperature overnight, and the solvent wasthen distilled off under reduced pressure. Ethyl acetate and water wereadded to the resulting residue, and the organic layer was washed withwater, a 10% aqueous solution of citric acid, a saturated aqueous sodiumhydrogen carbonate, and brine, and dried over sodium sulfate. Theresultant was distillated under reduced pressure, and the resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=90:10 (v/v) to 50:50 (v/v)] to afford the desired compound (2.11g, 82%).

MS (APCI, ESI) m/z: 435 (M+H)⁺

Step 2: Compound 12-3

The compound obtained in step 1 (2.11 g, 4.85 mmol) was reacted in thesame manner as in step 1 of Example 10, except that L-isoleucine wasused in place of N⁶-[(2,2,2-trichloroethoxy)carbonyl]-L-lysinehydrochloride, to afford the desired compound (2.16 g, 99%).

MS (APCI, ESI) m/z: 451 (M+H)⁺

Step 3: Compound 12-4

The compound obtained in step 2 (2.16 g, 4.85 mmol) was reacted in thesame manner as in step 2 of Example 5 to afford the desired compound(1.48 g, 56%).

Step 4: Compound 12-5

The compound obtained in step 3 (1.48 g, 2.67 mmol) was reacted in thesame manner as in step 3 of Example 10 to afford the desired compound(0.794 g, 89%).

Step 5: Compound 12-6

The compound obtained in step 4 (0.794 g, 2.38 mmol) was reacted in thesame manner as in step 1 of Example 7 to afford the desired compound(0.886 g, 89%).

Step 6: Compound 12-7

The compound obtained in step 5 (0.794 g, 2.12 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(1.19 g, 72%).

MS (APCI, ESI) m/z: 1006 (M+H)⁺

Step 7: Compound 12-8

The compound obtained in step 6 (1.19 g, 1.18 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.07 g, quantitative).

MS (APCI, ESI) m/z: 892 (M+H)⁺

Step 8: Compound 12-9

The compound obtained in step 7 (1.18 mmol) was reacted in the samemanner as in step 8 of Example 1 to afford the desired compound (0.800g, 76%).

MS (APCI, ESI) m/z: 890 (M+H)⁺

Step 9: Compound 12-10

The compound obtained in step 8 (0.800 g, 0.899 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.567 g, 90%).

MS (APCI, ESI) m/z: 1004 (M+H)⁺

Step 10: Compound 12-11

The compound obtained in step 9 (0.567 g, 0.564 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.454 g, 94%).

MS (APCI, ESI) m/z: 848 (M+H)⁺

Step 11: Compound 12-12

The compound obtained in step 10 (0.100 g, 0.118 mmol) was reacted inthe same manner as in step 9 of Example 4 to afford the desired compound(0.159 g, quantitative).

MS (APCI, ESI) m/z: 1324 (M+H)⁺

Step 12: Compound 12-13

The compound obtained in step 11 (0.118 mmol) was reacted in the samemanner as in step 11 of Example 3 to afford the desired compound (0.139g, 97%).

MS (APCI, ESI) m/z: 1210 (M+H)⁺

Step 13: Compound 12-14

The compound obtained in step 12 (0.139 g, 0.114 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0667 g, 56%).

¹H-NMR (CDCl₃) δ: 8.81 (1H, s), 8.21-8.19 (1H, m), 7.55-7.44 (4H, m),7.33-7.31 (2H, m), 7.22 (1H, s), 7.13-7.11 (2H, m), 6.90-6.87 (2H, m),6.39 (1H, s), 6.11 (1H, s), 5.89-5.87 (1H, m), 5.35-5.32 (1H, m),4.80-4.58 (2H, m), 4.30 (1H, s), 4.22-4.07 (5H, m), 3.89 (3H, s),3.81-3.72 (9H, m), 3.58-3.53 (3H, m), 3.38-3.31 (2H, m), 2.98-2.93 (2H,m), 2.76-2.72 (1H, m), 2.42-2.39 (1H, m), 2.18-2.12 (3H, m), 1.94-1.51(7H, m), 1.31-1.13 (1H, m), 0.97-0.90 (6H, m), 0.71-0.66 (4H, m).

MS (APCI, ESI) m/z: 1042 (M+H)⁺

Step 14: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-D-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-isoleucinamide

The compound obtained in step 13 (0.0314 g, 0.0301 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0300 g, 69%).

MS (APCI, ESI) m/z: 1443 (M+H)⁺

Example 13: Drug-Linker 11

Step 1: [2-(2-{[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}ethoxy)ethoxy] acetate

To a solution of starting material 13-1 (3.00 g, 7.78 mmol, TokyoChemical Industry Co., Ltd.) in N,N-dimethylformamide (10 mL),1,8-diazabicyclo[5.4.0]-7-undecene (1.16 mL, 7.78 mL) was added, and theresultant was stirred at room temperature for 2 hours, and1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidin-2,5-dione (1.10 g, 2.72 mmol) andtriethylamine (1.94 mL, 14.0 mmol) was then added thereto at roomtemperature. The reaction solution was distillated under reducedpressure, and the resulting residue was then purified by silica gelcolumn chromatography [chloroform:methanol=100:0 (v/v) tochloroform:methanol=90:10 (v/v)] to afford the desired compound (0.410g, 12%).

¹H-NMR (CDCl₃) δ: 7.68-7.66 (1H, m), 7.55-7.53 (1H, m), 7.44-7.24 (6H,m), 6.58-6.56 (1H, m), 5.16-5.12 (1H, m), 4.16-4.11 (2H, m), 3.80-3.57(5H, m), 3.48-3.44 (2H, m), 3.30-3.18 (2H, m), 2.90-2.86 (1H, m),2.52-2.45 (1H, m), 2.26-2.22 (1H, m), 2.02-1.98 (1H, m).

MS (APCI, ESI) m/z: 451 (M+H)⁺

Step 2: N-{[2-(2-{[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}ethoxy)ethoxy]acetyl}-L-valyl-N-{4-[({[(I1a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-1′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 1 (0.050 g, 0.0499 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0590 g, 82%).

MS (APCI, ESI) m/z: 1434 (M+H)⁺

Example 14: Drug-Linker 12

Step 1: N-[20-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-16,20-dioxo-4,7,10,13-tetraoxa-17-azaicosan-1-oyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

A solution of the compound obtained in step 11 of Example 4 (0.0500 g,0.0499 mmol), starting material 14-1 (0.050 g, 0.0499 mmol, commerciallyavailable from Alfa Aesar), and triethylamine (0.00830 mL, 0.0599 mmol)in dichloromethane (3 mL) was stirred at room temperature overnight. Theresultant was distillated under reduced pressure, and the resultingresidue was then purified by silica gel column chromatography[chloroform:methanol=100:0 (v/v) to chloroform:methanol=90:10 (v/v)] toafford the desired compound (0.0490 g, 64%).

MS (APCI, ESI) m/z: 1536 (M+H)⁺

Example 15: Drug-Linker 13

Step 1:Dimethyl(6S,6′S)-5,5′-{1,5-pentanediylbis[oxy(5-methoxy-2-nitrobenzen-4,1-diyl)carbonyl]}bis(5-azaspiro[2.4]heptane-6-carboxylate)

To a solution of starting material 15-1 (5.41 g, 10.9 mmol, Journal ofMedicinal Chemistry 2004, 47, 1161) in dichloromethane (50 mL), oxalylchloride (5.63 mL, 65.7 mmol) was added at 0° C., andN,N-dimethylformamide (0.0844 mL, 1.09 mmol) was added dropwise thereto.The temperature of the reaction solution was raised to room temperature,and the reaction solution was stirred for 2 hours. The resultant wasdistillated under reduced pressure, and the resulting residue wasdissolved in dichloromethane (100 mL), which was added dropwise todichloromethane solution (100 mL) of methyl(6S)-5-azaspiro[2.4]heptane-6-carboxylate hydrochloride (4.28 g, 24.1mmol, Tetrahedron Letters 2012. 53. 3847) and triethylamine (6.07 mL,43.8 mmol) under the nitrogen atmosphere at −40° C. The temperature ofthe reaction solution was raised to 0° C., and the reaction solution wasstirred for 2 hours. To the reaction mixture, 1 N hydrochloric acid (100mL) was added, and the organic layer was washed with water and brine,and dried over anhydrous sodium sulfate. The resultant was distillatedunder reduced pressure to afford the desired compound (8.40 g,quantitative).

¹H-NMR (DMSO-D₆) δ: 7.71 (2H, s), 6.88 (2H, s), 4.63 (2H, m), 4.15-4.12(4H, m), 3.94 (6H, s), 3.71 (6H, s), 3.25 (2H, m), 3.10 (2H, m),2.31-2.28 (2H, m), 1.90-1.83 (6H, m), 1.60-1.58 (2H, m), 0.71-0.49 (8H,m).

MS (APCI, ESI) m/z: 769 (M+H)⁺.

Step 2: {1,5-Pentanediylbis[oxy(5-methoxy-2-nitrobenzen-4,1-diyl)]}bis{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]methanone}

To a solution of the compound obtained in step 1 (8.40 g, 10.9 mmol) inTHF (100 mL), lithium borohydride (714 mg, 32.8 mmol) was added, and theresultant was stirred at 0° C. for 30 minutes, and the temperature wasraised to room temperature, and stirring was performed for 1 hour. After1 N hydrochloric acid was added at 0° C., the resultant was extractedwith ethyl acetate, and washed with brine, and then dried over anhydroussodium sulfate. The solvent was distilled off under reduced pressure toafford the desired compound (7.70 g, 99%).

¹H-NMR (DMSO-D₆) δ: 7.67 (2H, s), 7.05 (2H, s), 4.86-4.74 (2H, m),4.22-4.12 (6H, m), 3.92 (6H, s), 3.83-3.73 (2H, m), 3.62-3.51 (2H, m),3.29 (1H, m), 3.11 (2H, m), 2.96 (1H, m), 2.12-2.03 (2H, m), 1.82-1.77(6H, m), 1.59-1.56 (2H, m), 0.67-0.41 (8H, m).

MS (APCI, ESI) m/z: 713 (M+H)⁺.

Step 3: Pentan-1,5-diylbis[oxy(5-methoxy-2-nitrobenzen-4,1-diyl)carbonyl(6S)-5-azaspiro[2.4]heptan-5,6-diylmethanediyl] diacetate

The compound obtained in step 2 (7.70 g, 10.8 mmol) was dissolved inpyridine (20 mL) and acetic anhydride (10 mL, 105.9 mmol), which wasstirred at room temperature. The resultant was distillated under reducedpressure to afford the desired compound (8.38 g, 97%).

¹H-NMR (DMSO-D₆) δ: 7.68 (2H, s), 7.03 (2H, s), 4.47-4.46 (2H, m),4.36-4.27 (4H, m), 4.13-4.11 (6H, m), 3.92 (6H, s), 3.16 (2H, m), 2.98(2H, m), 2.17 (1H, m), 2.06 (6H, s), 1.84 (4H, m), 1.68 (1H, m), 1.58(2H, m), 0.64-0.45 (8H, m).

MS (APCI, ESI) m/z: 797 (M+H)⁺.

Step 4: 1,5-Pentanediylbis[oxy(2-amino-5-methoxybenzen-4,1-diyl)carbonyl(6S)-5-azaspiro[2.4]heptan-5,6-diylmethanediyl] diacetate

To a solution of the compound obtained in step 3 (8.28 g, 10.4 mmol) inN,N-dimethylformamide (100 mL), 5% palladium carbon (moisture content:54%, 1.00 g) was added, and the reaction solution was then vigorouslystirred under the hydrogen atmosphere at room temperature for 6 hours.The resultant was filtered through a Celite, and the filtrate was thendistillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [chloroform:methanol=100:0(v/v) to 90:10 (v/v)] to afford the desired compound (5.05 g, 66%).

¹H-NMR (DMSO-D₆) δ: 6.66 (2H, s), 6.36 (2H, s), 5.11 (4H, s), 4.49 (2H,s), 4.19 (4H, m), 3.90 (4H, m), 3.62 (6H, s), 3.48-3.46 (2H, m), 3.33(2H, s), 3.23-3.20 (2H, m), 2.01 (6H, s), 1.78-1.74 (6H, m), 1.55 (2H,m), 0.61-0.58 (4H, m), 0.49-0.48 (4H, m).

MS (APCI, ESI) m/z: 737 (M+H)⁺.

Step 5:{(6S)-5-[4-({5-[4-({(6S)-6-[(Acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-5-amino-2-methoxyphenoxy]pentyl}oxy)-5-methoxy-2-{[(prop-2-en-1-yloxy)carbonyl]amino}benzoyl]-5-azaspiro[2,4]hept-6-yl}methylacetate (monoallyloxycarbonyl form)

To a solution of the compound obtained in step 4 (5.05 g, 6.85 mmol) indichloromethane (100 mL), pyridine (1.10 mL, 13.7 mmol) was added, andallyl chloroformate (0.725 mL, 6.85 mmol) was added thereto under thenitrogen atmosphere at −78° C., and the resultant was stirred for 2hours. The resultant was distillated under reduced pressure, and theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=70:30 (v/v) to 100:0 (v/v),chloroform:methanol=100:0 (v/v) to 90:10 (v/v)] to afford thebisallyloxycarbonyl form (1.36 g, 22%) and monoallyloxycarbonyl form(2.63 g, 47%) as the desired compound.

Pentan-1,5-diylbis[oxy(5-methoxy-2-{[(prop-2-en-1-yloxy)carbonyl]amino}benzen-4,1-diyl)carbonyl(6S)-5-azaspiro[2.4]heptan-5,6-diylmethanediyl] diacetate(bisallyloxycarbonyl form):

¹H-NMR (DMSO-D₆) δ: 9.14 (2H, s), 7.14 (2H, s), 6.85 (2H, s), 5.94 (2H,m), 5.33 (2H, m), 5.21 (2H, m), 4.55 (4H, m), 4.47 (1H, s), 4.23 (3H,s), 3.96 (4H, m), 3.74 (6H, s), 3.34 (6H, s), 3.31 (2H, m), 3.21 (2H,m), 2.04 (6H, s), 1.79 (4H, m), 1.67 (2H, m), 1.56 (2H, m), 0.56-0.48(8H, m).

MS (APCI, ESI) m/z: 905 (M+H)⁺.

Monoallyloxycarbonyl form:

¹H-NMR (DMSO-D₆) δ: 9.14 (1H, s), 7.14 (1H, s), 6.85 (1H, s), 6.65 (1H,s), 6.35 (1H, s), 5.95 (1H, m), 5.33 (1H, m), 5.22 (1H, m), 5.11 (2H,s), 4.55 (2H, m), 4.48 (2H, s), 4.23-4.14 (4H, m), 3.96 (2H, m), 3.90(2H, m), 3.74 (3H, s), 3.63 (3H, s), 3.49 (1H, m), 3.38-3.30 (4H, m),3.21 (1H, m), 2.04 (3H, s), 2.01 (3H, s), 1.77 (5H, m), 1.68 (1H, m),1.56 (2H, m), 0.63-0.48 (8H, m).

MS (APCI, ESI) m/z: 821 (M+H)⁺.

Step 6:N-[(2-Propen-1-yloxy)carbonyl]-L-valyl-N-{4-[({[2-({(6S)-6-[(acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-5-({5-[4-({(6S)-6-[(acetyloxy)methyl]-5-azaspiro[2.4]hept-5-yl}carbonyl)-2-methoxy-5-{[(2-propen-1-yloxy)carbonyl]amino}phenoxy]pentyl}oxy)-4-methoxyphenyl]carbamoyl}oxy)methyl]phenyl}-L-alaninamide

The monoallyloxycarbonyl form obtained in step 5 (2.00 g, 2.44 mmol) wasreacted in the same manner as in step 6 of Example 1 to afford thedesired compound (2.64 g, 89%).

¹H-NMR (DMSO-D₆) δ: 10.02 (1H, s), 9.14 (2H, s), 8.18 (1H, m), 7.59 (2H,m), 7.33 (2H, m), 7.27 (1H, m), 7.14 (2H, s), 6.85 (2H, s), 5.99-5.86(2H, m), 5.31 (2H, m), 5.19 (2H, m), 5.03 (2H, s), 4.55 (2H, m), 4.48(2H, m), 4.41 (2H, m), 4.23-4.21 (3H, m), 3.94-3.91 (4H, m), 3.88-3.86(2H, m), 3.74 (3H, s), 3.74 (3H, s), 3.34 (4H, s), 3.32-3.30 (2H, m),3.20-3.18 (2H, m), 2.03 (6H, s), 1.96 (1H, m), 1.79 (4H, s), 1.66 (1H,m), 1.55 (2H, s), 1.30 (3H, m), 0.88 (3H, m), 0.83 (3H, m), 0.54-0.49(8H, m).

MS (APCI, ESI) m/z: 1224 (M+H)⁺.

Step 7:N-[(2-Propen-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-5-{[5-(4-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-2-methoxy-5-{[(2-propen-1-yloxy)carbonyl]amino}phenoxy)pentyl]oxy}-4-methoxyphenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

To a solution of the compound obtained in step 6 (2.64 g, 2.16 mmol) inmethanol (10 mL), potassium carbonate (1.49 g, 10.8 mmol) was added, andthe resultant was stirred at room temperature for 3 hours. A saturatedaqueous ammonium chloride (100 mL) was added to the reaction mixture,which was extracted with ethyl acetate. The organic layer was dried overanhydrous sodium sulfate. The resultant was distillated under reducedpressure to afford the desired compound (2.21 g, 90%).

¹H-NMR (DMSO-D₆) δ: 10.04 (1H, s), 9.18 (1H, s), 8.18 (1H, m), 7.59 (2H,m), 7.33 (2H, m), 7.26 (1H, m), 7.22 (1H, s),7.14 (2H, s), 6.89 (2H, s),5.98-5.86 (2H, m), 5.31 (2H, m), 5.19 (2H, m), 5.04 (2H, s), 4.80 (2H,m), 4.55 (2H, m), 4.48 (2H, m), 4.4 1 (1H, m), 4.26 (2H, s), 3.96-3.94(4H, m), 3.90-3.85 (1H, m), 3.74 (6H, s), 3.59 (2H, m), 3.33 (6H, s),3.09 (1H, m), 1.93-1.83 (8H, m), 1.57-1.54 (2H, m), 1.30 (3H, m), 0.88(3H, m), 0.83 (3H, m), 0.52-0.43 (8H, m).

MS (APCI, ESI) m/z: 1140 (M+H)⁺.

Step 8:N-[(2-Propen-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-{[5-({(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-10′-[(2-propen-1-yloxy)carbonyl]-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl}oxy)pentyl]oxy}-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 7 (2.03 g, 1.78 mmol) indichloromethane (50 mL), Dess-Martin periodinane (1.59 g, 3.74 mmol) wasadded, and the resultant was stirred at room temperature overnight. Asaturated aqueous sodium hydrogen carbonate (100 mL) was added to thereaction mixture, which was extracted with chloroform. The organic layerwas dried over anhydrous sodium sulfate. The resultant was distillatedunder reduced pressure, and the resulting residue was purified by silicagel column chromatography [chloroform:methanol=100:0 (v/v) to 90:10(v/v)] to afford the desired compound (2.05 g, quantitative).

¹H-NMR (DMSO-D₆) δ: 9.99 (1H, s), 8.16 (1H, m), 7.54 (2H, m), 7.32-7.22(3H, m), 7.08-7.04 (2H, m), 6.80-6.72 (2H, m), 6.55 (2H, s), 5.94-5.86(2H, m), 5.75 (2H, m), 5.31-5.04 (2H, m), 4.81 (1H, m), 4.62 (1H, m),4.48-4.38 (4H, m), 4.00-3.87 (4H, m), 3.79-3.76 (7H, m), 3.54 (2H, m),3.42-3.40 (2H, m), 3.33 (4H, s), 3.14 (2H, m), 2.35 (2H, m), 1.80-1.78(4H, m), 1.59-1.56 (4H, m), 1.29 (3H, m), 0.87 (3H, m), 0.83 (3H, m),0.70-0.59 (8H, m).

MS (APCI, ESI) m/z: 1136 (M+H)⁺.

Step 9:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 8 (2.05 g, 1.80 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(1.02 g, 60%).

¹H-NMR (DMSO-D₆) δ: 10.08 (1H, s), 7.57 (2H, m), 7.32-7.20 (3H, m), 7.05(2H, s), 6.68-6.60 (3H, m), 5.74 (1H, m), 4.99-4.58 (4H, m), 3.99-3.94(4H, m), 3.78-3.73 (6H, m), 3.66-3.38 (4H, m), 3.15-3.01 (3H, m),2.40-2.34 (3H, m), 1.89-1.81 (6H, m), 1.57-1.53 (4H, m), 1.28 (3H, m),0.88 (3H, m), 0.78 (3H, m), 0.64-0.55 (8H, m).

MS (APCI, ESI) m/z: 950 (M+H)⁺.

Step 10: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 9 (0.710 g, 0.747 mmol) and the compoundobtained in step 1 of Example 2 (0.313 g, 0.747 mmol) were dissolved inmixed solvent of dichloromethane (1.5 mL) and methanol (0.1 mL).Thereto, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (0.264 g, 0.897 mmol) was added, and the resultant was stirredat room temperature for 1 hour. The resultant was distillated underreduced pressure, and the resulting residue was purified by silica gelcolumn chromatography [chloroform:methanol=100:0 (v/v) to 80:20 (v/v)]to afford the desired compound (0.671 g, 66%).

Table 2: Peak positions of proton NMR and MS for drug-linker 13

¹H-NMR (DMSO-D₆) δ: 9.91 (1H, s), 8.32 (1H, s), 8.23-7.91 (3H, m),7.81-7.19 (14H, m), 7.04 (1H, m), 6.80-6.62 (3H, m), 5.77-5.75 (1H, m),5.20 (1H, m), 5.01 (1H, m), 4.79 (1H, m), 4.46-4.35 (1H, m), 4.04 (4H,m), 3.86-3.38 (18H, m), 3.22-3.15 (2H, m), 2.67-2.63 (1H, m), 2.46-2.23(3H, m), 2.09-1.91 (2H, m), 1.80-1.78 (5H, m), 1.57 (3H, m), 1.27 (3H,s), 1.11-1.04 (1H, m), 0.87-0.79 (6H, m), 0.63-0.55 (6H, m).

MS (APCI, ESI) m/z: 1351 (M+H)⁺.

Example 16: Drug-Linker 14

Step 1: N-[6-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 9 of Example 15 (0.100 g, 0.105 mmol) andazadibenzocyclooctynoic acid (0.0351 g, 0.105 mmol) were reacted in thesame manner as in step 10 of Example 15 to afford the desired compound(0.0702 g, 53%).

¹H-NMR (DMSO-D₆) δ: 9.92 (1H, s), 8.14 (1H, m), 7.92-7.19 (16H, m), 7.04(1H, m), 6.86-6.72 (1H, m), 6.60-6.58 (1H, m), 5.76 (1H, m), 5.20 (1H,m), 5.03 (1H, m), 4.81-4.78 (1H, m), 4.43-4.37 (2H, m), 4.11-3.41 (14H,m), 3.21-3.15 (3H, m), 2.43-2.37 (2H, m), 2.19-2.15 (1H, m), 2.03-1.92(3H, m), 1.77-1.75 (5H, m), 1.55 (4H, s), 1.26-1.18 (6H, m), 1.09-1.04(1H, m), 0.87-0.77 (6H, m), 0.69-0.50 (8H, m).

MS (APCI, ESI) m/z: 1265 (M+H)⁺.

Example 17: Drug-Linker 15

Step 1: Compound 17-2

Starting raw material 17-1 (2.00 g, 2.81 mmol, WO 2013053872) and2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (2.00g, 8.51 mmol) were reacted in the same manner as in step 6 of Example 3to afford the desired compound (1.89 g, quantitative).

MS (APCI, ESI) m/z: 672 (M+H)⁺.

Step 2: Compound 17-3

The compound obtained in step 1 (1.89 g, 2.81 mmol) was dissolved in amixed solvent of ethanol (30 mL and formic acid (1.5 mL). Zinc powder(3.68 g) was added thereto, and the resultant was stirred at roomtemperature for 1 hour. The resultant was filtered through a Celite, anda saturated aqueous sodium hydrogen carbonate (100 mL) was added to thefiltrate, which was extracted with ethyl acetate. The organic layer wasdried over anhydrous sodium sulfate. The resultant was distillated underreduced pressure to afford the desired compound (1.81 g, quantitative).

MS (APCI, ESI) m/z: 642 (M+H)⁺.

Step 3: Compound 17-4

The compound obtained in step 2 (1.81 g, 2.82 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(1.76 g, 86%).

MS (APCI, ESI) m/z: 726 (M+H)⁺.

Step 4: Compound 17-5

The compound obtained in step 3 (1.76 g, 2.42 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(1.05 g, 71%).

MS (APCI, ESI) m/z: 612 (M+H)⁺.

Step 5: Compound 17-6

The compound obtained in step 4 (1.05 g, 1.71 mmol) was reacted in thesame manner as in step 3 of Example 9 to afford the desired compound(0.686 g, 66%).

MS (APCI, ESI) m/z: 610 (M+H)⁺.

Step 6: Compound 17-7

The compound obtained in step 5 (0.481 g, 0.789 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.288 g, 72%).

MS (APCI, ESI) m/z: 508 (M+H)⁺.

Step 7: Compound 17-8

The compound obtained in step 6 (0.288 g, 0.567 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.268 g, 93%).

MS (APCI, ESI) m/z: 510 (M+H)⁺.

Step 8: Compound 17-9

The compound obtained in step 7 (0.267 g, 0.525 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.278 g, 89%).

MS (APCI, ESI) m/z: 594 (M+H)⁺.

Step 9: Compound 17-10

The compound obtained in step 8 (0.278 g, 0.468 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.207 g, quantitative).

MS (APCI, ESI) m/z: 438 (M+H)⁺.

Step 10: Compound 17-11

Using the compound obtained in step 11 of Example 1 (0.307 g, 0.331mmol), the compound obtained in step 9 (0.0964 g, 0.220 mmol) wasreacted in the same manner as in step 10 of Example 3 to afford thedesired compound (0.224 g, 79%).

MS (APCI, ESI) m/z: 1285 (M+H)⁺.

Step 11: Compound 17-12

The compound obtained in step 10 (0.294 g, 0.228 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.284 g, quantitative).

MS (APCI, ESI) m/z: 1171 (M+H)⁺.

Step 12: Compound 17-13

The compound obtained in step 11 (0.284 g, 0.242 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.114 g, 47%).

MS (APCI, ESI) m/z: 1003 (M+H)⁺.

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[7-methoxy-2-(6-methoxy-3-pyridinyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 12 (0.114 g, 0.113 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0121 g, 8%).

¹H-NMR (CDCl₃) δ: 8.75 (1H, s), 8.24-8.09 (2H, m), 7.85-6.98 (13H, m),6.75-6.73 (2H, m), 6.57-6.47 (1H, m), 6.18 (1H, s), 5.89 (1H, s),5.37-4.96 (3H, m), 4.67-4.60 (3H, m), 4.41-4.06 (6H, m), 3.92 (3H, s),3.86-3.82 (3H, m), 3.74-3.70 (3H, m), 3.59-3.45 (3H, m), 3.32-3.23 (2H,m), 2.81-2.64 (3H, m), 2.28-2.04 (4H, m), 1.49-1.38 (4H, m), 1.23-1.22(2H, m), 1.09-1.01 (3H, m), 0.96-0.90 (5H, m), 0.69-0.64 (6H, m).

MS (APCI, ESI) m/z: 1404 (M+H)⁺.

Example 18: Drug-Linker 16

Step 1: Compound 18-1

Starting raw material 17-1 (2.00 g, 2.81 mmol) and2-methyl-5-pyridinylboronic acid (1.00 g, 7.30 mmol) were used andsubjected to Suzuki-Miyaura coupling reaction in the same manner as instep 6 of Example 3 to afford the desired compound (0.901 g, 49%).

MS (APCI, ESI) m/z: 656 (M+H)⁺.

Step 2: Compound 18-2

The compound obtained in step 1 (1.98 g, 3.02 mmol) was reacted in thesame manner as in step 2 of Example 17 to afford the desired compound(1.86 g, 98%).

MS (APCI, ESI) m/z: 626 (M+H)⁺.

Step 3: Compound 18-3

The compound obtained in step 2 (1.86 g, 2.97 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(1.36 g, 65%).

MS (APCI, ESI) m/z: 710 (M+H)⁺.

Step 4: Compound 18-4

The compound obtained in step 3 (1.36 g, 2.42 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(0.991 g, 87%).

MS (APCI, ESI) m/z: 596 (M+H)⁺.

Step 5: Compound 18-5

The compound obtained in step 4 (0.991 g, 1.66 mmol) was reacted in thesame manner as in step 3 of Example 9 to afford the desired compound(0.608 g, 62%).

MS (APCI, ESI) m/z: 594 (M+H)⁺.

Step 6: Compound 18-6

The compound obtained in step 5 (0.405 g, 0.682 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.239 g, 71%).

¹H-NMR (DMSO-D₆) δ: 8.60 (1H, s), 8.02 (1H, m), 7.87 (1H, m), 7.67 (1H,s), 7.62 (1H, m), 7.57-7.54 (1H, m), 7.40 (1H, s), 7.25 (1H, m), 6.74(1H, s), 4.53-4.49 (1H, m), 3.85 (3H, s), 3.52 (2H, m), 2.46 (3H, s),1.30-1.24 (3H, m), 1.07-1.06 (18H, m), observed as a water adduct of thedesired compound.

MS (APCI, ESI) m/z: 492 (M+H)⁺.

Step 7: Compound 18-7

The compound obtained in step 6 (0.239 g, 0.485 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.180 g, 75%).

MS (APCI, ESI) m/z: 494 (M+H)⁺.

Step 8: Compound 18-8

The compound obtained in step 7 (0.180 g, 0.364 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.179 g, 85%).

MS (APCI, ESI) m/z: 578 (M+H)⁺.

Step 9: Compound 18-9

The compound obtained in step 8 (0.179 g, 0.309 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.137 g, quantitative).

MS (APCI, ESI) m/z: 422 (M+H)⁺.

Step 10: Compound 18-10

The compound obtained in step 9 (0.0780 g, 0.185 mmol) was reacted inthe same manner as in step 10 of Example 3, except that the compoundobtained in step 11 of Example 1 (0.258 g, 0.278 mmol) was used in placeof the compound obtained in step 10 of Example 1, to afford the desiredcompound (0.213 g, 91%).

MS (APCI, ESI) m/z: 1269 (M+H)⁺.

Step 11: Compound 18-11

The compound obtained in step 10 (0.213 g, 0.168 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.182 g, 94%).

¹H-NMR (DMSO-D₆) δ: 10.00-9.85 (1H, m), 8.54 (1H, s), 8.32 (1H, m), 8.16(1H, s), 7.82 (1H, m), 7.65-7.56 (3H, m), 7.35-7.06 (6H, m), 6.79 (1H,m), 6.57 (1H, s), 5.87-5.80 (3H, m), 5.26-5.09 (5H, m), 4.85-4.83 (1H,m), 4.56-4.40 (5H, m), 4.13 (5H, m), 3.92-3.87 (2H, m), 3.80 (5H, s),3.58-3.54 (1H, m), 3.21-3.09 (3H, m), 2.81 (1H, m), 2.45 (3H, s),2.36-2.34 (1H, m), 2.16-2.10 (2H, m), 1.98-1.92 (1H, m), 1.57 (2H, m),1.30-1.28 (4H, m), 0.92-0.84 (7H, m), 0.67-0.62 (4H, m).

MS (APCI, ESI) m/z: 1155 (M+H)⁺.

Step 12: Compound 18-12

The compound obtained in step 11 (0.182 g, 0.157 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0751 g, 48%).

¹H-NMR (DMSO-D₆) δ: 10.15 (1H, s), 8.54 (1H, m), 8.50 (1H, s), 7.78 (1H,m), 7.67 (1H, s), 7.57 (2H, m), 7.30 (1H, s),7.21-7.19 (3H, m), 7.05(1H, s), 6.77 (1H, s), 6.60-6.56 (2H, m), 6.35 (1H, s), 5.91-5.83 (2H,m), 5.76 (1H, m), 5.29-5.13 (4H, m), 4.84 (1H, m), 4.54-4.49 (1H, m),4.19-4.03 (4H, m), 3.79 (3H, s), 3.65 (3H, s), 3.57-3.53 (2H, m),3.42-3.40 (1H, m), 3.28-3.26 (1H, m), 3.14 (1H, m), 2.81 (1H, m), 2.44(3H, s), 2.37-2.33 (1H, m), 2.21-2.01 (3H, m), 1.57 (1H, m), 1.30-1.26(3H, m), 0.93-0.83 (6H, m), 0.67-0.61 (4H, m).

MS (APCI, ESI) m/z: 987 (M+H)⁺.

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[7-methoxy-2-(6-methyl-3-pyridinyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 12 (0.0751 g, 0.0761 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0117 g, 11%).

¹H-NMR (CDCl₃) δ: 8.75 (1H, s), 8.48-8.46 (1H, m), 7.69-7.56 (4H, m),7.47-6.89 (14H, m), 6.45 (1H, m), 6.24-6.19 (1H, m), 5.91 (1H, m), 5.37(1H, m), 5.01 (1H, m), 4.67-4.59 (3H, m), 4.30-3.19 (22H, m), 2.88-2.63(3H, m), 2.55 (3H, s), 2.42-2.34 (2H, m), 2.27-2.04 (4H, m), 1.51-1.34(4H, m), 1.12-1.06 (3H, m), 0.99-0.84 (3H, m), 0.71-0.66 (4H, m).

MS (APCI, ESI) m/z: 1388 (M+H)⁺.

Example 19: Drug-Linker 17

Step 1:[(2S)-2-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-4-(4-methoxyphenyl)-2,3-dihydro-1H-pyrrol-1-yl](5-methoxy-2-nitro-4-{[tri(propan-2-yl)silyl]oxy}phenyl)methanone

Starting material 17-1 (2.00 g, 2.81 mmol) was reacted in the samemanner as in step 6 of Example 3 to afford the desired compound (1.31 g,93%).

¹H-NMR (CDCl₃) δ: 7.75-7.73 (1H, m), 7.12 (2H, m), 6.82-6.76 (4H, m),6.13-6.11 (1H, m), 4.80-4.70 (1H, m), 3.93-3.91 (3H, m), 3.79-3.75 (4H,m), 3.21-3.15 (1H, m), 3.01-2.93 (1H, m), 1.34-1.25 (3H, m), 1.12 (18H,m), 0.89 (9H, s), 0.13-−0.18 (6H, m).

MS (APCI, ESI) m/z: 671 (M+H)⁺

Step 2:(2-Amino-5-methoxy-4-{[tri(propan-2-yl)silyl]oxy}phenyl)[(2S)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(4-methoxyphenyl)-2,3-dihydro-1H-pyrrol-1-yl]methanone

The compound obtained in step 1 (1.31 g, 1.95 mmol) was reacted in thesame manner as in step 2 of Example 17 to afford the desired compound(1.12 g, 90%).

¹H-NMR (CDCl₃) δ: 7.21-7.18 (2H, m), 6.85-6.81 (2H, m), 6.79-6.76 (2H,m), 6.28 (1H, s), 4.42 (2H, m), 3.98-3.93 (1H, m), 3.90-3.86 (1H, m),3.80 (3H, s), 3.71 (3H, s), 3.11 (1H, m), 2.98 (1H, m), 1.32-1.23 (4H,m), 1.12-1.10 (18H, m), 0.85 (9H, s), 0.08-0.02 (6H, m).

Step 3: Prop-2-en-1-yl(2-{[(2S)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(4-methoxyphenyl)-2,3-dihydro-1H-pyrrol-1-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamate

The compound obtained in step 2 (1.12 g, 1.59 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.890 g, 77%).

¹H-NMR (CDCl₃) δ: 8.57 (1H, m), 7.77 (1H, m), 7.18 (2H, m), 6.86-6.78(4H, m), 5.95-5.90 (1H, m), 5.32 (1H, m), 5.20 (1H, m), 4.79-4.77 (1H,m), 4.64-4.57 (2H, m), 4.00-3.98 (1H, m), 3.93-3.91 (1H, m), 3.80 (3H,s), 3.76 (3H, s), 3.14-3.09 (1H, m), 3.00 (1H, m), 1.36-1.25 (3H, m),1.14-1.11 (18H, m), 0.85 (9H, s), 0.11-0.03 (6H, m).

MS (APCI, ESI) m/z: 725 (M+H)⁺

Step 4: Prop-2-en-1-yl(2-{[(2S)-2-(hydroxymethyl)-4-(4-methoxyphenyl)-2,3-dihydro-1H-pyrrol-1-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamate

The compound obtained in step 3 (0.890 g, 1.23 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(0.696 g, 93%).

¹H-NMR (CDCl₃) δ: 8.54-8.36 (1H, m), 7.71 (1H, m), 7.18-7.17 (2H, m),6.86-6.84 (3H, m), 6.77 (1H, m), 5.94-5.90 (1H, m), 5.32 (1H, m), 5.21(1H, m), 4.87-4.85 (1H, m), 4.61 (2H, m), 4.50 (1H, m), 3.96-3.84 (2H,m), 3.80 (3H, s), 3.76 (3H, s), 3.28 (1H, m), 2.64 (1H, m), 1.36-1.25(3H, m), 1.13 (18H, m).

MS (APCI, ESI) m/z: 611 (M+H)⁺

Step 5: Prop-2-en-1-yl(11aS)-11-hydroxy-7-methoxy-2-(4-methoxyphenyl)-5-oxo-8-{[tri(propan-2-yl)silyl]oxy}-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 4 (0.696 g, 1.14 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(0.532 g, 77%).

¹H-NMR (CDCl₃) δ: 7.36 (1H, s), 7.31-7.29 (2H, m), 7.22 (1H, s),6.90-6.87 (2H, m), 6.72 (1H, m), 5.82-5.76 (2H, m), 5.19-5.14 (2H, m),4.60 (1H, m), 4.49-4.46 (1H, m), 3.98-3.96 (1H, m), 3.86 (3H, s), 3.82(3H, s), 3.44 (1H, m), 3.36 (1H, m), 3.05 (1H, m), 1.28-1.21 (3H, m),1.10-1.07 (18H, m).

MS (APCI, ESI) m/z: 609 (M+H)⁺

Step 6: Prop-2-en-1-yl(11aS)-11-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-2-(4-methoxyphenyl)-5-oxo-8-{[tri(propan-2-yl)silyl]oxy}-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 5 (0.532 g, 0.874 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.532 g, 95%).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.29 (2H, m), 7.23 (1H, s), 6.89 (2H,m), 6.70 (1H, s), 5.90 (1H, m), 5.76 (1H, m), 5.14-5.10 (2H, m), 4.60(1H, m), 4.38 (1H, m), 3.93-3.85 (1H, m), 3.87 (3H, s), 3.82 (3H, s),3.32 (1H, m), 2.82-2.78 (1H, m), 1.29-1.22 (3H, m), 1.12-1.07 (18H, m),0.89 (9H, s), 0.27 (3H, s), 0.20 (3H, s).

MS (APCI, ESI) m/z: 723 (M+H)⁺

Step 7: Prop-2-en-1-yl(11aS)-11-{[tert-butyl(dimethyl)silyl]oxy}-8-hydroxy-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 6 (0.532 g, 0.756 mmol) was reacted likestep 10 of Example 1 to afford the desired compound (0.359 g, 86%).

¹H-NMR (CDCl₃) δ: 7.34 (1H, s), 7.30-7.27 (3H, m), 6.90-6.88 (2H, m),6.76 (1H, s), 5.93-5.90 (2H, m), 5.81-5.73 (1H, m), 5.12-5.08 (2H, m),4.61 (1H, m), 4.42 (1H, m), 3.97 (3H, s), 3.93-3.88 (1H, m), 3.83 (3H,s), 3.31 (1H, m), 2.83-2.79 (1H, m), 0.91 (9H, s), 0.27 (3H, s), 0.22(3H, s).

MS (APCI, ESI) m/z: 567 (M+H)⁺

Step 8: Prop-2-en-1-yl(11aS)-8-(3-bromopropoxy)-11-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 7 (0.405 g, 0.715 mmol) was reacted in thesame manner as in step 1 of Example 4 to afford the desired compound(0.490 g, 99%).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.29 (2H, m), 6.89 (2H, m), 6.69 (1H,s), 5.94 (1H, m), 5.82-5.75 (1H, m), 5.13-5.08 (1H, m), 5.13-5.08 (2H,m), 4.65 (1H, m), 4.41 (1H, m), 4.20-4.13 (2H, m), 3.94-3.88 (1H, m),3.92 (3H, s), 3.83 (3H, s), 3.62 (2H, m), 3.32 (1H, m), 2.83-2.80 (1H,m), 2.41-2.36 (2H, m), 0.91 (9H, s), 0.27 (3H, s), 0.24 (3H, s).

MS (APCI, ESI) m/z: 687 (M+H)⁺

Step 9:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-8′-[3-({(11aS)-11-{[tert-butyl(dimethyl)silyl]oxy}-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-11′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 8 (0.490 g, 0.713 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.600 g, 60%).

MS (APCI, ESI) m/z: 1414 (M+H)⁺

Step 10:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-[3-({(11aS)-11-hydroxy-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 9 (0.600 g, 0.424 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.500 g, 99%).

MS (APCI, ESI) m/z: 1184 (M−H)⁺

Step 11:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 9 (0.500 g, 0.421 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.113 g, 27%).

¹H-NMR (CDCl₃) δ: 8.94 (1H, s), 7.70 (1H, s), 7.56-7.53 (2H, m),7.46-7.44 (2H, m), 7.36 (1H, s), 7.31 (2H, m), 7.23 (1H, s), 7.03 (2H,m), 6.90-6.88 (2H, m), 6.68 (1H, m), 6.57 (1H, s), 6.40 (1H, s), 5.92(1H, m), 5.43 (1H, m), 4.67 (1H, m), 4.55-4.53 (1H, m), 4.46 (1H, m),4.35-4.33 (1H, m), 4.28-4.24 (1H, m), 4.15-4.13 (1H, m), 3.88 (3H, s),3.87 (3H, s), 3.83 (3H, s), 3.77-3.72 (1H, m), 3.62-3.60 (1H, m),3.52-3.47 (2H, m), 3.34 (1H, m), 3.30-3.28 (1H, m), 3.00-2.91 (2H, m),2.50-2.41 (2H, m), 2.24-2.22 (1H, m), 2.10-2.08 (1H, m), 1.77-1.75 (1H,m), 1.40-1.37 (1H, m), 1.16 (3H, m), 0.82 (3H, m), 0.76-0.62 (4H, m),0.69 (3H, m).

MS (APCI, ESI) m/z: 1000 (M+H)⁺

Step 12: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 11 (0.157 g, 0.157 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.120 g, 49%).

MS (APCI, ESI) m/z: 1401 (M+H)⁺

Example 20: Drug-Linker 18

Step 1: {Propan-1,3-diylbis[oxy(5-methoxy-2-nitrobenzen-4,1-diyl)]}bis{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]methanone}

Starting raw material 20-1 (3.00 g, 6.43 mmol, Journal of the AmericanChemical Society 1992, 13, 4939) and the compound obtained in step 3 ofExample 1 (3.42 g, 14.2 mmol) were reacted in the same manner as in step1 of Example 15 to afford the desired compound (3.74 g, 64%).

¹H-NMR (CDCl₃) δ: 7.79-7.70 (2H, m), 6.83-6.75 (2H, m), 4.52-4.50 (1.5H,m), 4.35-4.29 (4.5H, m), 4.03 (0.5H, m), 3.97-3.92 (6H, m), 3.88 (0.5H,m), 3.60-3.52 (1H, m), 3.38-3.33 (0.5H, m), 3.26-3.24 (0.5H, m),3.04-2.93 (3H, m), 2.45-2.39 (2H, m), 2.25-2.21 (1H, m), 2.09-1.98 (1H,m), 1.68 (1H, m), 1.56 (1H, m), 0.93-0.90 (14H, m), 0.77-0.74 (4H, m),0.71-0.62 (4H, m), 0.57-0.49 (4H, m), 0.44-0.40 (2H, m), 0.11 (9H,m),−0.14 (3H, m).

MS (APCI, ESI) m/z: 912 (M+H)⁺

Step 2: {Propan-1,3-diylbis[oxy(2-amino-5-methoxybenzen-4,1-diyl)]}bis{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]methanone}}

The compound obtained in step 1 (3.74 g, 4.10 mmol) was reacted in thesame manner as in step 4 of Example 15 to afford the desired compound(2.97 g, 85%).

¹H-NMR (CDCl₃) δ: 7.79-7.69 (2H, m), 6.82-6.75 (2H, m), 4.54-4.47 (1.5H,m), 4.36-4.26 (4.5H, m), 4.03 (0.5H, m), 3.98-3.92 (6H, m), 3.88 (0.5H,m), 3.61-3.51 (1H, m), 3.39-3.32 (0.5H, m), 3.28-3.21 (0.5H, m),3.05-2.93 (3H, m), 2.45-2.39 (2H, m), 2.24-2.21 (1H, m), 2.08-2.06 (1H,m), 2.00-1.99 (1H, m), 1.69-1.66 (1H, m), 1.57-1.54 (5H, m), 0.94-0.88(14H, m), 0.78-0.74 (4H, m), 0.71-0.62 (4H, m), 0.57-0.49 (4H, m),0.44-0.40 (2H, m), 0.13-0.10 (9H, m), −0.11-−0.17 (3H, m).

MS (APCI, ESI) m/z: 853 (M+H)⁺

Step 3: Prop-2-en-1-yl(5-[3-(5-amino-4-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-2-methoxyphenyl)propoxy]-2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxyphenyl)carbamate

The compound obtained in step 2 (2.97 g, 3.48 mmol) was reacted in thesame manner as in step 5 of Example 15 to afford the desired compound(0.549 g, 17%).

¹H-NMR (CDCl₃) δ: 9.18 (1H, m), 7.88 (1H, m), 6.80 (1H, m), 6.73 (1H,s), 6.31 (1H, s), 5.96 (1H, m), 5.36 (1H, m), 5.24 (1H, m), 4.68-4.59(4H, m), 4.59-4.43 (2H, m), 4.27-4.25 (2H, m), 4.20-4.18 (2H, m), 4.00(2H, m), 3.79-3.72 (9H, m), 3.05 (1H, m), 2.35 (2H, m), 2.32-2.19 (2H,m), 1.78-1.50 (4H, m), 0.99-0.89 (20H, m), 0.67-0.54 (4H, m), 0.50-0.48(2H, m), 0.05 (12H, m).

MS (APCI, ESI) m/z: 1021 (M+H)⁺

Step 4:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-5-[3-(4-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-2-methoxy-5-{[(prop-2-en-1-yloxy)carbonyl]amino}phenoxy)propoxy]-4-methoxyphenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 3 (0.549 g, 0.586 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(0.402 g, 51%).

MS (APCI, ESI) m/z: 1341 (M+H)⁺

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-[4-({[(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-5-[3-(4-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-2-methoxy-5-{[(prop-2-en-1-yloxy)carbonyl]amino}phenoxy)propoxy]-4-methoxyphenyl)carbamoyl]oxy}methyl)phenyl]-L-alaninamide

The compound obtained in step 4 (0.402 g, 0.300 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(0.282 g, 85%).

MS (A PCI, ESI) m/z: 1120 (M+H)⁺

Step 6:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-[3-({(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-10′-[(prop-2-en-1-yloxy)carbonyl]-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl}oxy)propoxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 5 (0.282 g, 0.253 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(0.0600 g, 21%).

MS (APCI, ESI) m/z: 1106 (M−H)⁺

Step 7:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 6 (0.0600 g, 0.0541 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0347 g, 70%).

MS (APCI, ESI) m/z: 922 (M+H)⁺

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.0347 g, 0.0376 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.00770 g, 16%).

MS (APCI, ESI) m/z: 1323 (M+H)⁺

Example 21: Drug-Linker 19

Step 1: Compound 21-2

To a solution of starting raw material 21-1 (11.8 g, 20.2 mmol, WO2013053872) and pyridine (1.79 mL, 22.2 mmol) in THF (50 mL), aceticanhydride (2.10 mL, 22.3 mmol) was slowly added under ice-cooling.Subsequently, 4-dimethylaminopyridine (0.459 g, 3.76 mmol) was addedthereto, and the resultant was stirred at room temperature. After theraw materials disappeared, water was added to the reaction mixture,which was extracted with ethyl acetate. The organic layer was washedwith water and brine, and dried over anhydrous sodium sulfate. Theresultant was filtered and then distillated under reduced pressure, andthe resulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=90:10 (v/v) to 60:40 (v/v)] to afford the desiredcompound (12.3 g, 97%).

MS (APCI, ESI) m/z: 625 (M+H)⁺

Step 2: Compound 21-3

The compound obtained in step 1 (12.3 g, 19.7 mmol) was reacted in thesame manner as in step 4 of Example 15 to afford the desired compound(11.3 g, 97%) MS (APCI, ESI) m/z: 595 (M+H)⁺

Step 3: Compound 21-4

The compound obtained in step 2 (11.3 g, 19.0 mmol) was reacted in thesame manner as in step 9 of Example 3, except that 2,2,2-trichloroethylchloroformate (2.93 mL, 21.9 mmol) was used in place of allylchloroformate, to afford the desired compound (12.4 g, 85%).

MS (APCI, ESI) m/z: 769 (M+H)⁺

Step 4: Compound 21-5

The compound obtained in step 3 (12.4 g, 16.1 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(9.90 g, 94%).

MS (APCI, ESI) m/z: 655 (M+H)⁺

Step 5: Compound 21-6

The compound obtained in step 4 (9.90 g, 15.1 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(8.19 g, 83%).

MS (APCI, ESI) m/z: 653 (M+H)⁺

Step 6: Compound 21-7

To the compound obtained in step 5 (3.00 g, 4.59 mmol) intetrahydrofuran (10 mL) and a 10% aqueous solution of ammonium acetate(10 mL), 10% Cd/Pb (3.00 g, 24.0 mmol, 90 mass %) was added, and theresultant was vigorously stirred under the nitrogen atmosphere. Afterthe raw materials disappeared, the reaction mixture was filtered. Thefiltrate was extracted with dichloromethane. The organic layer waswashed with brine, and dried over anhydrous sodium sulfate. Theresultant was filtered, and then distillated under reduced pressure, andthe resulting compound (2.10 g, 99%) was directly used for thesubsequent reaction.

MS (APCI, ESI) m/z: 461 (M+H)⁺

Step 7: Compound 21-8

The compound obtained in step 6 (2.10 g, 4.56 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(2.09 g, 99%).

MS (APCI, ESI) m/z: 463 (M+H)⁺

Step 8: Compound 21-9

The compound obtained in step 7 (2.09 g, 4.52 mmol) was reacted in thesame manner as in step 3 of Example 21 to afford the desired compound(2.88 g, 100%).

¹H-NMR (CDCl₃) δ: 7.23 (1H, s), 6.81 (1H, s), 5.40-5.37 (1H, m), 4.95(1H, m), 4.41 (1H, m), 4.21 (1H, m), 4.05 (1H, m), 3.96-3.92 (1H, m),3.86 (3H, s), 3.79-3.75 (1H, m), 3.64 (1H, m), 2.34-2.28 (1H, m),2.18-2.13 (1H, m), 2.05 (3H, s), 1.30-1.19 (3H, m), 1.11-1.04 (18H, m).

MS (APCI, ESI) m/z: 637 (M+H)⁺

Step 9: Compound 21-10

To a mixed solution of the compound obtained in step 8 (2.28 g, 4.51mmol) in methanol (15 mL) and tetrahydrofuran (5 mL), a solution ofpotassium carbonate (0.624 g, 4.52 mmol) in water (15 mL) was slowlyadded dropwise, and the resultant was stirred at room temperature. Afterthe raw materials disappeared, water was added to the reaction mixture,which was extracted with ethyl acetate. The organic layer was washedwith brine, and dried over anhydrous sodium sulfate. The resultant wasfiltered, and then distillated under reduced pressure, and the resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=90:10 (v/v) to 0:100 (v/v)] to afford the desired compound (2.08g, 77%).

¹H-NMR (CDCl₃) δ: 7.18 (1H, s), 6.80 (1H, s), 4.96 (1H, m), 4.64-4.59(1H, m), 4.40 (1H, m), 4.18 (1H, m), 4.00-3.92 (2H, m), 3.82 (3H, s),3.65 (2H, m), 2.28-2.20 (2H, m), 2.04-1.97 (1H, m), 1.27-1.20 (3H, m),1.09-1.05 (18H, m).

MS (APCI, ESI) m/z: 595 (M+H)⁺

Step 10: Compound 21-11

To a solution of the compound obtained in step 9 (2.08 g, 3.49 mmol) and2,2,6,6-tetramethyl-1-piperidyloxy radical (0.109 g, 0.698 mmol) indichloromethane (50 mL), iodobenzene diacetate (2.00 g, 6.21 mmol) wasslowly added under ice-cooling. The reaction mixture was stirred at roomtemperature for 2 hours. After the raw materials disappeared, water wasadded to the reaction mixture, and the reaction mixture was extractedwith dichloromethane. The organic layer was washed with water and brine,and dried over anhydrous magnesium sulfate. The resultant was filtered,and then distillated under reduced pressure, and the resulting residuewas purified by silica gel column chromatography [hexane:ethylacetate=90:10 (v/v) to 60:40 (v/v)] to afford the desired compound (1.94g, 94%).

¹H-NMR (CDCl₃) δ: 7.21 (1H, s), 6.84 (1H, s), 4.96 (1H, m), 4.44 (1H,m), 4.34-4.23 (3H, m), 3.99-3.92 (1H, m), 3.86 (3H, s), 3.68-3.62 (1H,m), 2.94 (1H, m), 2.50-2.46 (1H, m), 1.29-1.21 (3H, m), 1.21-1.21 (18H,m).

MS (APCI, ESI) m/z: 593 (M+H)⁺

Step 11: Compound 21-12

The compound obtained in step 10 (1.94 g, 3.27 mmol) was reacted in thesame manner as in step 5 of Example 3 to afford the desired compound(2.17 g, 92%).

¹H-NMR (CDCl₃) δ: 7.22-7.18 (2H, m), 6.84 (1H, s), 4.95 (1H, m),4.45-4.39 (2H, m), 4.23-4.15 (1H, m), 3.85 (3H, s), 3.64 (1H, m),3.36-3.30 (1H, m), 2.74-2.68 (1H, m), 1.29-1.19 (3H, m), 1.11-1.04 (18H,m).

Step 12: Compound 21-13

The compound obtained in step 11 (0.837 g, 1.15 mmol) andquinoxaline-6-boronic acid pinacol ester (1.18 g, 4.61 mmol) werereacted in the same manner as in step 6 of Example 3 to afford thedesired compound (0.713 g, 88%).

¹H-NMR (CDCl₃) δ: 8.82-8.77 (2H, m), 8.05 (1H, m), 7.95 (1H, m),7.79-7.75 (2H, m), 7.25 (1H, s), 6.88 (1H, s), 4.96 (1H, m), 4.49-4.40(2H, m), 4.37-4.28 (1H, m), 3.88 (3H, s), 3.75 (1H, m), 3.48 (1H, m),2.90 (1H, m), 1.30-1.22 (3H, m), 1.13-1.06 (18H, m).

MS (APCI, ESI) m/z: 705 (M+H)⁺

Step 13: Compound 21-14

The compound obtained in step 12 (0.713 g, 1.01 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.400 g, 72%).

¹H-NMR (CDCl₃) δ: 8.81-8.80 (2H, m), 8.05 (1H, m), 7.95 (1H, m),7.80-7.75 (2H, m), 7.29 (1H, s), 6.96 (1H, s), 6.10 (1H, m), 5.07 (1H,m), 4.45-4.32 (3H, m), 3.98 (3H, s), 3.80-3.73 (1H, m), 3.51-3.46 (1H,m), 2.91 (1H, m).

MS (APCI, ESI) m/z: 549 (M+H)⁺

Step 14: Compound 21-15

Using the compound obtained in step 11 of Example 1 (0.321 g, 0.346mmol), the compound obtained in step 13 (0.200 g, 0.364 mmol) wassubjected to coupling reaction in the same manner as in step 10 ofExample 3 to afford the desired compound (0.475 g, 99%).

¹H-NMR (CDCl₃) δ: 8.83-8.79 (2H, m), 8.65-8.55 (1H, m), 8.09-7.98 (2H,m), 7.92-7.82 (2H, m), 7.47-7.31 (2H, m), 7.24-7.19 (1H, m), 7.14-7.02(2H, m), 6.97-6.88 (1H, m), 6.80-6.66 (1H, m), 6.55-6.47 (1H, m),6.06-6.00 (1H, m), 5.97-5.85 (1H, m), 5.51-5.09 (3H, m), 4.82-4.71 (2H,m), 4.63-4.52 (2H, m), 4.48-4.30 (2H, m), 4.26-4.17 (2H, m), 4.16-4.09(1H, m), 4.08-3.98 (3H, m), 3.9-3.73 (6H, m), 3.53-3.44 (2H, m),3.28-3.26 (1H, m), 2.94-2.91 (1H, m), 2.40-2.33 (2H, m), 2.21-2.13 (1H,m), 2.07-2.03 (4H, m), 1.67-1.50 (2H, m), 1.46-1.39 (2H, m), 1.29-1.24(2H, m), 1.00-0.60 (18H, m), 0.22-−0.06 (6H, m).

MS (APCI, ESI) m/z: 1395 (M+H)⁺

Step 15: Compound 21-16

The compound obtained in step 14 (0.475 g, 0.340 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.310 g, 71%).

¹H-NMR (CDCl₃) δ: 8.83-8.79 (2H, m), 8.72 (1H, m), 8.07-8.01 (2H, m),7.88 (2H, m), 7.41-7.39 (2H, m), 7.23 (1H, m), 7.13 (2H, m), 6.84 (1H,m), 6.56 (1H, s), 5.95-5.88 (2H, m), 5.48-5.47 (1H, m), 5.32-5.10 (3H,m), 4.87-4.72 (2H, m), 4.61-4.55 (2H, m), 4.47-4.20 (3H, m), 4.07-4.03(2H, m), 3.90 (3H, s), 3.83 (3H, s), 3.80-3.72 (3H, m), 3.58 (1H, m),3.49 (1H, m), 3.31 (1H, m), 2.92 (1H, m), 2.41 (1H, m), 2.36-2.29 (1H,m), 2.19-2.11 (1H, m), 1.77-1.72 (1H, m), 1.68-1.66 (3H, m), 1.65-1.63(1H, m), 1.42-1.41 (3H, m), 0.97 (3H, m), 0.93 (3H, m), 0.76-0.61 (4H,m).

MS (APCI, ESI) m/z: 1282 (M+H)⁺

Step 16: Compound 21-17

The compound obtained in step 15 (0.310 g, 0.242 mmol) was reacted inthe same manner as in step 6 of Example 21 to afford the desiredcompound (0.168 g, 63%).

¹H-NMR (CDCl₃) δ: 8.85-8.76 (3H, m), 8.04-7.99 (3H, m), 7.86 (1H, s),7.49-7.41 (3H, m), 7.25-7.06 (3H, m), 6.96-6.83 (1H, m), 6.49 (1H, m),6.13 (1H, s), 5.51-5.45 (2H, m), 5.34-5.28 (2H, m), 5.21 (1H, m),4.80-4.37 (4H, m), 4.17-4.02 (6H, m), 3.88 (3H, s), 3.84-3.70 (6H, m),3.68-3.50 (5H, m), 3.31 (1H, m), 2.93-2.90 (1H, m), 2.42 (1H, m),2.29-2.12 (3H, m), 1.78-1.75 (1H, m), 1.44 (3H, m), 0.97 (3H, m), 0.94(3H, m), 0.79-0.60 (4H, m).

MS (APCI, ESI) m/z: 1108 (M+H)⁺

Step 17: Compound 21-18

The compound obtained in step 16 (0.168 g, 0.152 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.112 g, 72%).

¹H-NMR (CDCl₃) δ: 9.18 (1H, m), 8.78 (2H, m), 8.04-8.02 (2H, m),7.95-7.93 (2H, m), 7.77 (1H, s), 7.50-7.44 (3H, m), 7.23-7.21 (1H, m),7.11 (2H, m), 6.44 (1H, m), 6.11 (1H, m), 5.90 (1H, m), 5.34 (1H, m),4.74-4.63 (3H, m), 4.42 (1H, m), 4.16-4.03 (3H, m), 3.89 (3H, s), 3.80(3H, s), 3.74-3.72 (1H, m), 3.65-3.51 (4H, m), 3.32-3.28 (3H, m), 2.92(1H, m), 2.41 (1H, m), 2.34-2.28 (1H, m), 2.20-2.18 (2H, m), 1.76 (4H,m), 1.43 (3H, m), 1.00 (3H, m), 0.84 (3H, m), 0.75-0.62 (4H, m).

MS (APCI, ESI) m/z: 1024 (M+H)⁺

Step 18: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-5-oxo-2-(quinoxaline-6-yl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 18 (0.112 g, 0.109 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.110 g, 71%).

MS (APCI, ESI) m/z: 1425 (M+H)⁺

Example 22: Drug-Linker 20

Step 1: Compound 22-1

The compound obtained in step 5 of Example 21 (5.11 g, 7.81 mmol) wasreacted in the same manner as in step 9 of Example 1 to afford thedesired compound (5.70 g, 95.0%).

MS (APCI, ESI) m/z: 767 (M+H)⁺

Step 2: Compound 22-2

The compound obtained in step 1 (5.70 g, 7.42 mmol) was reacted in thesame manner as in step 9 of Example 21 to afford the desired compound(5.07 g, 94%).

MS (APCI, ESI) m/z: 725 (M+H)⁺

Step 3: Compound 22-3

The compound obtained in step 2 (5.07 g, 6.98 mmol) was reacted in thesame manner as in step 10 of Example 21 to afford the desired compound(4.44 g, 88%).

MS (APCI, ESI) m/z: 723 (M+H)⁺

Step 4: Compound 22-4

The compound obtained in step 3 (4.44 g, 6.13 mmol) was reacted in thesame manner as in step 5 of Example 3 to afford the desired compound(4.85 g, 92%).

¹H-NMR (CDCl₃) δ: 7.24-7.16 (1H, m), 6.78 (1H, s), 5.92 (1H, m), 5.05(1H, m), 4.34 (1H, m), 3.91-3.87 (2H, m), 3.86 (3H, s), 3.35-3.29 (1H,m), 2.80 (1H, m), 1.28-1.22 (3H, m), 1.10-1.05 (18H, m), 0.86 (9H, s),0.28 (3H, s), 0.21 (3H, s).

Step 5: Compound 22-5

The compound obtained in step 4 (1.20 g, 1.40 mmol) and6-methoxy-2-naphthylboronic acid (0.850 g, 4.21 mmol) were used andreacted in the same manner as in step 6 of Example 3 to afford thedesired compound (1.06 g, 88%).

¹H-NMR (CDCl₃) δ: 7.72-7.69 (2H, m), 7.59-7.51 (3H, m), 7.30 (1H, s),7.16-7.07 (2H, m), 6.82 (1H, s), 5.94 (1H, m), 5.06 (1H, m), 4.34 (1H,m), 3.99-3.95 (1H, m), 3.93 (3H, s), 3.88 (3H, s), 3.46 (1H, m), 2.94(1H, m), 1.30-1.23 (3H, m), 1.12-1.07 (18H, m), 0.93 (9H, s), 0.31 (3H,s), 0.23 (3H, s).

Step 6: Compound 22-6

The compound obtained in step 5 (1.06 g, 1.23 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.6126 g, 71%).

MS (APCI, ESI) m/z: 707 (M+H)⁺

Step 7: Compound 22-7

The compound obtained in step 6 (0.205 g, 0.290 mmol) and the compoundobtained in step 11 of Example 1 (0.255 g, 0.274 mmol) were subjected tocoupling reaction in the same manner as in step 10 of Example 3 toafford the desired compound (0.375 g, 83%).

¹H-NMR (CDCl₃) δ: 8.68 (1H, s), 7.72 (2H, m), 7.67-7.55 (3H, m),7.36-7.21 (4H, m), 7.16-7.06 (4H, m), 6.82-6.79 (2H, m), 6.53 (1H, s),6.03-6.02 (1H, m), 5.97-5.89 (2H, m), 5.36-5.30 (2H, m), 5.23-5.16 (3H,m), 4.83-4.80 (1H, m), 4.75-4.72 (1H, m), 4.61-4.55 (3H, m), 4.33-4.29(1H, m), 4.17-4.11 (2H, m), 4.06-4.01 (2H, m), 3.94 (3H, s), 3.92-3.90(2H, m), 3.81 (3H, s), 3.72-3.70 (1H, m), 3.51-3.47 (2H, m), 3.26 (1H,m), 2.99-2.95 (1H, m), 2.42-2.32 (2H, m), 2.20-2.13 (1H, m), 1.55-1.40(4H, m), 0.97-0.92 (18H, m), 0.84-0.81 (9H, m), 0.69-0.63 (4H, m),0.30-0.05 (12H, m).

Step 8: Compound 22-8

The compound obtained in step 7 (0.375 g, 0.241 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.236 g, 74%).

¹H-NMR (CDCl₃) δ: 8.70 (1H, s), 7.72-7.68 (3H, m), 7.63-7.58 (2H, m),7.43-7.41 (2H, m), 7.27-7.22 (2H, m), 7.16-7.12 (2H, m), 6.91-6.86 (2H,m), 6.56 (1H, s), 5.95-5.84 (2H, m), 5.49 (1H, m), 5.34-5.14 (4H, m),4.78 (1H, m), 4.64-4.53 (4H, m), 4.27-4.24 (2H, m), 4.17-4.02 (3H, m),3.97-3.88 (2H, m), 3.93 (3H, s), 3.89 (3H, s), 3.88 (3H, s), 3.75-3.72(2H, m), 3.61-3.48 (3H, m), 3.33-3.30 (2H, m), 3.23-3.19 (1H, m),2.44-2.39 (2H, m), 2.29-2.27 (2H, m), 2.17-2.11 (1H, m), 1.76-1.72 (1H,m), 1.43 (3H, m), 0.95 (3H, m), 0.92 (3H, m), 0.77-0.61 (4H, m).

Step 9: Compound 22-9

The compound obtained in step 8 (0.236 g, 0.178 mmol) was reacted in thesame manner as in step 6 of Example 21 to afford the desired compound(0.201 g, 99%).

MS (APCI, ESI) m/z: 1134 (M+H)⁺

Step 10: Compound 22-10

The compound obtained in step 9 (0.201 g, 0.177 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.180 g, 97%).

¹H-NMR (CDCl₃) δ: 9.17 (1H, s), 7.90 (1H, s), 7.72-7.68 (2H, m),7.63-7.53 (4H, m), 7.44-7.42 (2H, m), 7.25-7.22 (1H, m), 7.16-7.12 (3H,m), 7.02-6.99 (2H, m), 6.76-6.74 (1H, m), 6.59 (1H, s), 6.41 (1H, s),5.94-5.87 (2H, m), 5.42 (1H, m), 4.66 (1H, m), 4.56-4.49 (2H, m),4.40-4.38 (1H, m), 4.29-4.24 (1H, m), 4.17-4.11 (1H, m), 3.93 (3H, s),3.89 (3H, s), 3.87 (3H, s), 3.85-3.70 (2H, m), 3.65-3.59 (2H, m),3.37-3.31 (2H, m), 3.06 (1H, m), 2.46-2.41 (2H, m), 2.20 (1H, m),2.10-2.06 (1H, m), 1.76-1.74 (2H, m), 1.17 (3H, m), 0.88-0.63 (4H, m),0.78 (3H, m), 0.67 (3H, m).

MS (APCI, ESI) m/z: 1050 (M+H)⁺

Step 11: Compound 22-11

The compound obtained in step 10 (0.0870 g, 0.0828 mmol) was reacted inthe same manner as in step 8 of Example 3 to afford the desired compound(0.0650 g, 75%).

¹H-NMR (CDCl₃) δ: 9.19 (1H, s), 7.92 (1H, m), 7.73-7.64 (4H, m),7.55-7.44 (4H, m), 7.22 (1H, s), 7.15-7.09 (4H, m), 6.43 (1H, s), 6.09(1H, s), 5.90 (2H, m), 5.34 (1H, m), 4.72 (1H, m), 4.65-4.63 (1H, m),4.36-4.34 (1H, m), 4.17-4.02 (4H, m), 3.92 (3H, s), 3.89 (3H, s), 3.80(3H, s), 3.78-3.72 (3H, m), 3.60-3.46 (5H, m), 3.31-3.27 (2H, m),2.89-2.85 (1H, m), 2.41 (1H, m), 2.33-2.26 (1H, m), 2.21-2.15 (2H, m),1.77-1.75 (1H, m), 1.43 (3H, m), 0.98 (3H, m), 0.83 (3H, m), 0.76-0.61(4H, m).

MS (APCI, ESI) m/z: 1051 (M+H)⁺

Step 12: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(1aS)-7-methoxy-2-(6-methoxynaphthalen-2-yl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 11 (0.065 g, 0.062 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.048 g, 54%).

MS (APCI, ESI) m/z: 1453 (M+H)⁺

Example 23: Drug-Linker 21

Step 1: Compound 23-2

Starting raw material 23-1 (14.8 g, 54.4 mmol, WO 2011130613) wasreacted in the same manner as in step 2 of Example 5 to afford thedesired compound (12.2 g, 63%).

MS (APCI, ESI) m/z: 379 (M+H)⁺

Step 2: Compound 23-3

The compound obtained in step 1 (3.78 g, 10.0 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(3.86 g, 40%).

MS (APCI, ESI) m/z: 967 (M+H)⁺

Step 3: Compound 23-4

The compound obtained in step 2 (3.860 g, 3.99 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(3.11 g, 92%).

¹H-NMR (CDCl₃) δ: 9.03 (1H, m), 8.70-8.63 (2H, m), 8.17-8.13 (1H, m),7.68 (1H, s), 7.35 (1H, m), 6.82-6.76 (2H, m), 5.93-5.83 (1H, m),5.49-5.42 (1H, m), 5.32-5.17 (4H, m), 4.73-4.52 (5H, m), 4.03 (1H, m),3.86 (1H, s), 3.80-3.75 (2H, m), 3.77 (3H, s), 3.65-3.63 (1H, m),3.12-3.10 (1H, m), 2.20-2.14 (1H, m), 1.93-1.88 (1H, m), 1.45 (3H, m),1.31-1.23 (3H, m), 1.10-1.08 (18H, m), 0.98 (3H, m), 0.94 (3H, m),0.64-0.47 (4H, m).

Step 4: Compound 23-5

The compound obtained in step 3 (3.11 g, 3.65 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(2.58 g, 84%).

¹H-NMR (CDCl₃) δ: 9.02-8.88 (1H, m), 8.69-8.59 (1H, m), 8.17-8.02 (1H,m), 7.22-7.17 (1H, m), 7.02 (1H, m), 6.79-6.78 (2H, m), 6.63 (1H, m),5.95-5.87 (2H, m), 5.33-5.11 (4H, m), 4.65-4.53 (3H, m), 4.01 (1H, m),3.83 (3H, s), 3.73 (1H, m), 3.59 (1H, m), 3.32 (1H, m), 2.43-2.39 (1H,m), 2.18-2.16 (1H, m), 1.75-1.67 (2H, m), 1.48-1.43 (3H, m), 1.20-1.14(3H, m), 1.10-0.94 (24H, m), 0.73-0.60 (4H, m).

Step 5: Compound 23-6

The compound obtained in step 4 (2.58 g, 3.03 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(2.75 g, 94%).

¹H-NMR (CDCl₃) δ: 8.78 (1H, m), 8.58 (1H, m), 8.03 (1H, m), 7.21 (1H,s), 6.97 (1H, m), 6.73 (1H, s), 6.59-6.55 (1H, m), 6.02 (1H, m),5.93-5.85 (1H, m), 5.32-5.04 (4H, m), 4.72-4.55 (3H, m), 3.99 (1H, m),3.84 (3H, s), 3.73-3.70 (1H, m), 3.53 (1H, m), 3.28 (1H, m), 2.36 (1H,m), 2.21-2.14 (1H, m), 1.55-1.53 (1H, m), 1.47-1.44 (4H, m), 1.23-1.16(3H, m), 1.10-1.00 (18H, m), 0.98 (3H, m), 0.94 (3H, m), 0.83 (9H, s),0.81-0.60 (4H, m), 0.21-0.19 (3H, m), 0.18-0.16 (3H, m).

Step 6: Compound 23-7

The compound obtained in step 5 (2.75 g, 2.85 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(2.28 g, 99%).

MS (APCI, ESI) m/z: 809 (M+H)⁺

Step 7: Compound 23-8

The compound obtained in step 6 (0.340 g, 0.420 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.530 g, 98%).

MS (APCI, ESI) m/z: 1285 (M+H)⁺

Step 8: Compound 23-9

The compound obtained in step 7 (0.530 g, 0.412 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.362 g, 75%).

MS (APCI, ESI) m/z: 1171 (M+H)⁺

Step 9: Compound 23-10

The compound obtained in step 8 (0.444 g, 0.379 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.347 g, 91%).

MS (APCI, ESI) m/z: 1103 (M+H)⁺

Step 10: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{6-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]pyridine-3-yl}-L-alaninamide

The compound obtained in step 9 (0.100 g, 0.0997 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.067 g, 48%).

MS (APCI, ESI) m/z: 1404 (M+H)⁺

Example 24: Drug-Linker 22

Step 1: Compound 24-2

To a suspension solution of methyltriphenylphosphonium bromide (7.28 g,20.4 mmol) in tetrahydrofuran (30 mL), potassium tert-butoxide (2.06 g,18.3 mmol) was added in small portions under the nitrogen atmosphere at0° C., and the resultant was then stirred for 2 hours. A solution ofcompound 24-1 (1.18 g, 2.04 mmol, WO 2013053872) in THF (10 mL) wasadded dropwise thereto over 2 minutes, and the resultant was stirred at0° C. for 28 hours. Water and an aqueous solution of citric acid wasadded to the reaction solution (pH=4.0), which was then extracted withethyl acetate. The organic layer was washed with brine, and dried oversodium sulfate. The resultant was distillated under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)] toafford the desired compound (0.450 g, 52%).

MS (APCI, ESI) m/z: 423 (M+H)⁺.

Step 2: Compound 24-3

To a solution of the compound obtained in step 1 (0.668 g, 1.58 mmol) inN,N-dimethylformamide (10 mL), imidazole (0.211 g, 3.16 mmol) was addedunder the nitrogen atmosphere. Thereafter, triisopropylsilyl chloride(0.502 mL, 2.37 mmol) was added dropwise thereto, and the resultant wasstirred at room temperature for 6 hours. To the reaction solution, a 10%aqueous solution of citric acid was added, which was twice extractedwith ethyl acetate. The organic layer was washed with brine, dried overanhydrous sodium sulfate, and then distillated under reduced pressure.The resulting residue was purified by silica gel chromatography[hexane:ethyl acetate=100:0 (v/v) to 50:50 (v/v)] to afford the desiredcompound (0.520 g, 57%).

MS (APCI, ESI) m/z: 579 (M+H)⁺.

Step 3: Compound 24-4

The compound obtained in step 2 (0.520 g, 0.898 mmol) was reacted in thesame manner as in step 2 of Example 17 to afford the desired compound(0.478 g, 97%).

MS (APCI, ESI) m/z: 547 (M−H)⁺.

Step 4: Compound 24-5

Under the nitrogen atmosphere, the compound obtained in step 3 (0.478 g,0.871 mmol) was reacted in the same manner as in step 9 of Example 3.After distillation under reduced pressure, the resulting residue (0.570g) was directly used for the subsequent reaction.

Step 5: Compound 24-6

The compound obtained in step 4 (0.570 g) was reacted in the same manneras in step 7 of Example 1 to afford the desired compound (0.446 g, 95%).

MS (APCI, ESI) m/z: 519 (M+H)⁺.

Step 6: Compound 24-7

The compound obtained in step 5 (0.446 g, 0.859 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(0.196 g, 44%).

¹H-NMR (CDCl₃) δ: 7.19 (1H, s), 6.68 (1H, s), 5.78-5.76 (1H, m), 5.55(1H, m), 5.19-5.13 (4H, m), 4.61-4.58 (1H, m), 4.49-4.46 (1H, m), 4.29(1H, m), 4.15 (1H, m), 3.85 (3H, s), 3.61 (1H, m), 3.38 (1H, s),2.93-2.90 (1H, m), 2.71 (1H, m), 1.30-1.18 (3H, m), 1.12-1.06 (18H, m).

MS (APCI, ESI) m/z: 517 (M+H)⁺.

Step 7: Compound 24-8

The compound obtained in step 6 (0.195 g, 0.377 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(0.230 g, 97%).

¹H-NMR (CDCl₃) δ: 7.19 (1H, s), 6.66 (1H, s), 5.75-5.65 (1H, m), 5.66(1H, m), 5.12-5.10 (4H, m), 4.59 (1H, m), 4.35 (1H, m), 4.28 (1H, m),4.11 (1H, m), 3.86 (3H, s), 3.53 (1H, m), 2.90-2.84 (1H, m), 2.48 (1H,m), 1.26-1.19 (3H, m), 1.09-1.06 (18H, m), 0.86 (9H, s), 0.23 (3H, s),0.17 (3H, s).

Step 8: Compound 24-9

The compound obtained in step 7 (0.230 g, 0.365 mmol) was reacted in thesame manner as in step 10 of Example 1. In this present step, a liquidseparation operation was performed after the completion of the reaction,and a crude product (0.238 g, quantitative) obtained by distilling offthe organic solvent under reduced pressure was used for the subsequentreaction.

Step 9: Compound 24-10

The compound obtained in the previous step and the compound obtained instep 10 of Example 1 (0.251 g, 0.311 mmol) were reacted in the samemanner as in step 2 of Example 3 to afford the desired compound (0.185g, 62%).

MS (APCI, ESI) m/z: 958 [⁸¹Br, (M+H)⁺], 956 [⁷⁹Br, (M+H)⁺].

Step 10: Compound 24-11

The compound obtained in step 8 (0.0660 g, 0.139 mmol) and the compoundobtained in step 9 (0.133 g, 0.139 mmol) were reacted in the same manneras in step 10 of Example 3 to afford the desired compound (0.123 g,66%).

MS (APCI, ESI) m/z: 1350 (M+H)⁺.

Step 11: Compound 24-12

The compound obtained in step 10 (0.123 g, 0.0910 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.0950 g, 92%).

¹H-NMR (CDCl₃) δ: 8.65 (1H, s), 7.59 (2H, m), 7.31-7.29 (2H, m),7.23-7.21 (3H, m), 6.89-6.86 (1H, m), 6.78 (1H, s), 6.38 (1H, s),5.92-5.88 (2H, m), 5.81-5.79 (1H, m), 5.61-5.52 (2H, m), 5.31 (2H, m),5.23 (1H, n),5.13-5.10 (4H, m), 5.05-5.02 (1H, m), 4.69-4.67 (2H, m),4.58-4.55 (2H, m), 4.49-4.46 (1H, m), 4.30 (2H, m), 4.16 (1H, m),3.98-3.96 (3H, m), 3.93 (3H, s), 3.89 (3H, s), 3.81-3.78 (2H, m), 3.74(1H, m), 3.69-3.66 (1H, m), 3.61 (1H, m), 3.43-3.41 (1H, m), 3.32 (1H,m), 2.89-2.87 (1H, m), 2.73 (1H, m), 2.42 (1H, m), 2.10-2.08 (1H, m),1.80-1.73 (4H, m), 1.53-1.50 (2H, m), 1.25-1.24 (3H, m), 0.93-0.89 (6H,m), 0.75-0.63 (4H, m).

MS (APCI, ESI) m/z: 1122 (M+H)⁺.

Step 12: Compound 24-13

The compound obtained in step 11 (0.0950 g, 0.0847 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0610 g, 76%).

¹H-NMR (CDCl₃) δ: 9.12-8.96 (1H, m), 7.82-7.60 (4H, m), 7.22-7.19 (3H,m), 6.94-6.66 (1H, m), 6.40-6.34 (2H, m), 5.89-5.86 (1H, m), 5.55 (1H,m), 5.40-5.07 (3H, m), 4.60-4.42 (4H, m), 4.23-4.09 (5H, m), 3.91-3.88(8H, m), 3.81-3.75 (8H, m), 3.60 (1H, m), 3.32-3.30 (2H, m), 3.24-3.22(1H, m), 3.12-3.09 (1H, m), 2.65-2.61 (1H, m), 2.41 (1H, m), 2.12-2.11(1H, m), 1.89-1.84 (9H, m), 1.75 (3H, m), 1.40-1.38 (2H, m), 1.25-1.21(3H, m), 0.99 (3H, m), 0.84 (3H, m), 0.74-0.66 (6H, m).

MS (APCI, ESI) m/z: 936 (M+H)⁺.

Step 13: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11aS)-7-methoxy-2-methylidene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 12 (0.0600 g, 0.0629 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.075 g, 89%).

¹H-NMR (DMSO-D₆) δ: 9.91 (1H, s), 8.21-8.16 (2H, m), 8.07-8.03 (1H, m),7.74-7.65 (3H, m), 7.60-7.44 (6H, m), 7.39-7.29 (5H, m), 7.21-7.19 (2H,m), 7.03 (1H, s), 6.85 (1H, s), 6.72 (1H, s), 6.58-6.56 (1H, m),5.77-5.74 (1H, m), 5.19-5.16 (3H, m), 5.03-5.00 (1H, m), 4.82-4.79 (1H,m), 4.36-4.33 (1H, m), 4.21-4.19 (1H, m), 4.14-4.11 (2H, m), 4.00-3.95(3H, m), 3.80-3.73 (101H, m), 3.65-3.52 (4H, m), 3.41-3.38 (2H, m),3.15-3.30 (1H, m), 3.14 (1H, m), 3.04-3.01 (1H, m), 2.68-2.66 (1H, m),2.32-2.28 (2H, m), 2.05-1.98 (2H, m), 1.78-1.77 (5H, m), 1.57-1.54 (3H,m), 1.28-1.25 (3H, m), 0.86-0.81 (6H, m), 0.67-0.62 (4H, m).

MS (APCI, ESI) m/z: 1337 (M+H)⁺.

Example 25: Drug-Linker 23

Step 1: Compound 25-1

Starting material 17-1 (3.81 g, 5.34 mmol) and tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)phenylcarbonate (5.13 g,16.0 mmol) were reacted in the same manner as in step 6 of Example 3 toafford the desired compound (3.05 g, 75%).

MS (APCI. ESI) m/z: 757 (M+H)⁺.

Step 2: Compound 25-2

The compound obtained in step 1 (3.05 g, 4.09 mmol) was reacted in thesame manner as in step 2 of Example 17 to afford the desired compound(2.67 g, 91%).

MS (APCI, ESI) m/z: 727 (M+H)⁺.

Step 3: Compound 25-3

The compound obtained in step 2 (2.67 g, 3.67 mmol) was reacted in thesame manner as in step 9 of Example 3. In this step, a liquid separationoperation was performed after the completion of the reaction, and acrude product (3.00 g, quantitative) obtained by concentrating theorganic solvent under reduced pressure was directly used for thesubsequent reaction.

Step 4: Compound 25-4

The compound obtained in step 3 (3.05 g) was reacted in the same manneras in step 7 of Example 1 to afford the desired compound (2.67 g,quantitative).

¹H-NMR (CDCl₃) δ: 8.34 (1H, m), 7.70 (1H, m), 7.24 (2H, m), 7.12 (2H,m), 6.86 (1H, s), 6.84 (1H, s), 5.95-5.89 (1H, m), 5.31 (1H, m), 5.21(1H, m), 4.87-4.86 (1H, m), 4.61-4.61 (2H, m), 4.41 (1H, m), 3.93-3.90(2H, m), 3.76 (3H, s), 3.29 (1H, m), 2.68-2.66 (1H, m), 1.55 (9H, s),1.33-1.26 (3H, m), 1.13-1.11 (18H, m).

MS (APCI, ESI) m/z: 697 (M+H)⁺.

Step 5: Compound 25-5

The compound obtained in step 4 (1.22 g, 1.75 mmol) was reacted in thesame manner as in step 3 of Example 9 to afford the desired compound(0.838 g, 69%).

¹H-NMR (CDCl₃) δ: 7.45 (1H, s), 7.37 (2H, m), 7.22 (1H, s), 7.15 (2H,m), 7.11-7.09 (1H, m), 6.72 (1H, s), 5.82-5.79 (1H, m), 5.19-5.16 (2H,m), 4.61-4.59 (2H, m), 4.50-4.47 (1H, m), 4.00 (1H, m), 3.86 (3H, s),3.41-3.28 (1H, m), 3.09-3.05 (1H, m), 1.57 (9H, s), 1.29-1.25 (3H, m),1.14-1.07 (18H, m).

MS (APCI, ESI) m/z: 695 (M+H)⁺.

Step 6: Compound 25-6

The compound obtained in step 5 (0.838 g, 1.21 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.745 g, quantitative).

¹H-NMR (CDCl₃) δ: 7.88 (1H, m), 7.50 (1H, s), 7.48-7.48 (1H, m), 7.40(2H, m), 7.18 (2H, m), 6.86 (1H, s), 4.45-4.43 (1H, m), 3.90 (3H, s),3.60-3.56 (1H, m), 3.38 (1H, m), 1.57 (9H, s), 1.31-1.26 (3H, m),1.11-1.10 (18H, m).

MS (APCI, ESI) m/z: 593 (M+H)⁺.

Steps 7 to 9: Compound 25-9

The compound obtained in step 6 (0.745 g, 1.26 mmol) was reacted in thesame manner as in steps 8 and 9 of Example 3 and step 10 of Example 1 toafford the desired compound (0.516 g, yields in three steps: 78%).

¹H-NMR (CDCl₃) δ: 7.46 (1H, s), 7.35 (2H, m), 7.27-7.25 (2H, m), 7.15(2H, m), 6.82 (1H, s), 5.93 (1H, s), 5.84-5.81 (1H, m), 5.12 (1H, m),4.61 (1H, m), 4.49-4.46 (1H, m), 4.35-4.32 (1H, m), 4.23-4.21 (1H, m),3.96 (3H, s), 3.65-3.62 (1H, m), 3.29 (1H, m), 2.71 (1H, m), 1.57 (9H,s).

MS (APCI, ESI) m/z: 523 (M+H)⁺.

Step 10: Compound 25-10

The compound obtained in step 9 (0.105 g, 0.200 mmol) and the compoundobtained in step 11 of Example 1 (0.186 g, 0.200 mmol) were subjected tocoupling reaction in the same manner as in step 10 of Example 3 toafford the desired compound (0.248 g, 90%).

¹H-NMR (CDCl₃) δ: 8.72-8.50 (1H, m), 7.52 (1H, s), 7.40-7.38 (4H, m),7.23-7.21 (2H, m), 7.16 (2H, m), 7.09 (1H, m), 6.84-6.82 (2H, m),6.51-6.49 (1H, m), 6.02 (1H, m), 5.92-5.90 (1H, m), 5.30-5.21 (5H, m),4.71-4.61 (6H, m), 4.36-4.24 (5H, m), 4.02-4.00 (3H, m), 3.93-3.88 (1H,m) 3.87 (3H, s), 3.81 (3H, s), 3.71 (2H, m), 3.51-3.49 (1H, m),3.32-3.28 (2H, m), 2.72 (1H, m), 2.37-2.34 (3H, m), 2.16-2.13 (1H, m),1.66 (9H, s), 1.50-1.46 (4H, m), 0.97-0.93 (7H, m), 0.82 (9H, s),0.68-0.65 (4H, m), 0.20 (3H, s), 0.14 (3H, s).

MS (APCI, ESI) m/z: 1370 (M+H)⁺.

Step 11: Compound 25-11

To a solution of the compound obtained in step 10 (0.248 g, 0.181 mmol)in dichloromethane (3 mL), piperidine (3 mL) was added, and theresultant was stirred at room temperature for 2 hours. The reactionsolution was diluted with an aqueous solution of citric acid, and twiceextracted with ethyl acetate. The organic layer was washed with brine,and dried over sodium sulfate. After filtration, the organic solvent wasdistilled off under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [chloroform:methanol=100:0(v/v) to 90:10 (v/v)] to afford the desired compound (0.153 g, 93%).

¹H-NMR (CDCl₃) δ: 8.77-8.69 (1H, m), 7.40-7.36 (3H, m), 7.23-7.21 (4H,m), 7.07 (2H, m), 6.88-6.86 (1H, m), 6.82 (2H, m), 6.51 (1H, s), 6.03(1H, m), 5.92-5.90 (1H, m), 5.30-5.21 (4H, m), 4.75-4.72 (2H, m),4.58-4.55 (3H, m), 4.37-4.35 (1H, m), 4.23-4.21 (3H, m), 4.01-3.99 (2H,m), 3.86 (3H, s), 3.79 (3H, s), 3.72 (2H, m), 3.66-3.64 (1H, m),3.51-3.48 (1H, m), 3.28 (2H, m), 2.67 (1H, m), 2.37-2.12 (4H, m),1.55-1.52 (2H, m), 1.45-1.42 (4H, m), 0.95-0.91 (6H, m), 0.81 (9H, s),0.81-0.78 (2H, m), 0.68-0.65 (3H, m), 0.20-0.15 (6H, m).

MS (APCI, ESI) m/z: 1270 (M+H)⁺.

Step 12: Compound 25-12

The compound obtained in step 11 (0.175 g, 0.138 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.162 g, quantitative).

MS (APCI, ESI) m/z: 1156 (M+H)⁺.

Step 13: Compound 25-13

The compound obtained in step 12 (0.116 g, 0.100 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0410 g, 41%).

MS (APCI, ESI) m/z: 988 (M+H)⁺.

Step 14: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-(3-{[(11aS)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 13 (0.00500 g, 0.00506 mmol) and thecompound obtained in step 2 of Example 2 (0.0100 g, 0.0202 mmol) weredissolved in dichloromethane (0.3 mL) and methanol (0.3 mL), andN,N-diisopropylethylamine (3.5 μL, 0.0202 mmol) was added thereto, andthe resultant was stirred at room temperature for 1 hour. The reactionsolution was distillated under reduced pressure, and the resultingresidue was purified by silica gel column chromatography[chloroform:CMW=100:0 (v/v) to 0:100 (v/v)] to afford the desiredcompound (0.00350 g, 50%).

MS (APCI, ESI) m/z: 1389 (M+H)⁺.

Example 26: Drug-Linker 24

Step 1: Compound 26-1

Starting material 17-1 (3.93 g, 5.51 mmol) and4-(hydroxymethyl)phenylboronic acid were reacted in the same manner asin step 6 of Example 3 to afford the desired compound (3.09 g, 84%).

MS (APCI, ESI) m/z: 671 (M+H)⁺.

Step 2: Compound 26-2

A solution of the compound obtained in step 1 (3.09 g, 4.61 mmol) indichloromethane (100 mL) was ice-cooled, to which triethylamine (1.60mL, 11.5 mmol) was added, and acetyl chloride (0.491 mL, 6.91 mmol) wasthen added dropwise thereto, and the resultant was stirred at roomtemperature for 2 hours. The reaction solution was diluted with anaqueous solution of citric acid, and three times extracted withchloroform. The organic layer was washed with a saturated aqueous sodiumcarbonate and brine, and dried over anhydrous sodium sulfate, and thesolvent was then distilled off under reduced pressure. The resultingcompound (3.75 g, quantitative) was directly used for the subsequentreaction.

Step 3: Compound 26-3

The compound obtained in step 2 (3.28 g, 4.60 mmol) was reacted in thesame manner as in step 2 of Example 17 to afford the desired compound(2.09 g, 67%).

MS (APCI, ESI) m/z: 682 (M+H)⁺.

Step 4: Compound 26-4

The compound obtained in step 3 (1.01 g, 1.48 mmol) was reacted in thesame manner as in step 9 of Example 3. The resultant was distillatedunder reduced pressure, and the resulting compound (1.19 g,quantitative) was directly used for the subsequent reaction.

Step 5: Compound 26-5

The compound obtained in step 4 (1.19 g, 1.55 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(0.885 g, 87%).

¹H-NMR (CDCl₃) δ: 8.34 (1H, m), 7.71 (1H, s), 7.31 (2H, m), 7.24 (2H,m), 6.91 (1H, s), 6.84 (1H, s), 5.97-5.88 (1H, m), 5.35-5.29 (1H, m),5.22-5.20 (1H, m), 5.07 (2H, s), 4.88-4.87 (1H, m), 4.62-4.61 (3H, m),4.33-4.31 (1H, m), 3.94-3.91 (2H, m), 3.76 (3H, s), 3.33-3.29 (1H, m),2.68 (1H, m), 1.33-1.29 (4H, m), 1.15-1.12 (18H, m).

MS (APCI, ESI) m/z: 653 (M+H)⁺.

Steps 6 and 7: Compound 26-7

The compound obtained in step 5 (0.885 g, 1.36 mmol) was treated in thesame manner as in step 3 of Example 9 and step 12 of Example 3 to affordthe desired compound (0.515 g, 85%).

MS (APCI, ESI) m/z: 549 (M+H)⁺.

Step 8-10: Compound 26-10

The compound obtained in step 7 (0.515 g, 0.983 mmol) was reacted in thesame manner as in steps 8 and 9 of Example 3 and step 10 of Example 1 toafford the desired compound (0.448 g, quantitative).

¹H-NMR (CDCl₃) δ: 7.51 (1H, s), 7.37-7.30 (4H, m), 7.26-7.25 (1H, m),6.82 (1H, s), 5.94 (1H, s), 5.86-5.79 (1H, m), 5.15-5.13 (1H, m), 5.09(3H, s), 4.61 (1H, m), 4.48-4.46 (1H, m), 4.35-4.32 (1H, m), 4.25-4.23(1H, m), 3.96 (3H, s), 3.64 (1H, d, m), 3.33-3.29 (1H, m), 2.73 (1H, m),2.11 (3H, s).

MS (APCI, ESI) m/z: 479 (M+H)⁺.

Step 11: Compound 26-11

The compound obtained in step 10 (0.0690 g, 0.144 mmol) and the compoundobtained in step 11 of Example 1 (0.134 g, 0.144 mmol) were reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.118 g, 62%).

MS (APCI, ESI) m/z: 1325 (M+H)⁺.

Steps 12 and 13: Compound 26-13

The compound obtained in step 11 (0.134 g, 0.101 mmol) was reacted inthe same manner as in steps 11 and 12 of Example 3 to afford the desiredcompound (0.0950 g, yield in two steps: 90%)

¹H-NMR (CDCl₃) δ: 9.14 (1H, s), 7.91 (1H, m), 7.69 (1H, s), 7.47 (4H,m), 7.40-7.38 (2H, m), 7.34-7.32 (2H, m), 7.21 (1H, s), 7.18 (2H, m),7.13 (2H, m), 6.40 (1H, s), 6.08 (1H, s), 5.88 (1H, m), 5.36 (1H, m),5.09 (2H, s), 4.72 (1H, m), 4.62-4.59 (2H, m), 4.35-4.32 (1H, m),4.12-4.07 (4H, m), 3.89 (3H, s), 3.80 (3H, s), 3.74-3.71 (2H, m),3.58-3.53 (3H, m), 3.41-3.38 (1H, m), 3.31-3.29 (2H, m), 2.78-2.74 (1H,m), 2.41 (1H, m), 2.31-2.31 (1H, m), 2.18-2.15 (1H, m), 2.11 (3H, s),1.76 (1H, m), 1.44-1.42 (3H, m), 0.99 (3H, m), 0.83 (3H, m), 0.71-0.66(4H, m).

MS (APCI, ESI) m/z: 1044 (M+H)⁺.

Step 14: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(1a′S)-8′-(3-{[(11aS)-2-{4-[(acetyloxy)methyl]phenyl}-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-11′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 13 (0.0700 g, 0.0670 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0520 g, 54%).

MS (APCI, ESI) m/z: 1445 (M+H)⁺.

Example 27: Drug-Linker 25

Step 1: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-[3-({(11aS)-2-[4-(hydroxymethyl)phenyl]-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

To a solution of the compound obtained in step 14 of Example 26 (0.0230g, 0.0159 mmol) in methanol (2 mL), 1 N sodium hydroxide solution(0.0175 mL, 0.0175 mmol) was added, and the resultant was stirred atroom temperature for 1 hour. Thereto, 1 N sodium hydroxide solution(0.0175 mL, 0.0175 mmol) was further added, and the resultant wasstirred at room temperature for 30 minutes. To the reaction solution, 1N hydrochloric acid aqueous solution (0.0350 mL) and water were added,and the resultant was three times extracted with chloroform. The organiclayer was dried over sodium sulfate, and distillated under reducedpressure, and the resulting residue was then purified by silica gelcolumn chromatography [chloroform:CMW=100:0 (v/v) to 0:100 (v/v)] toafford the desired compound (0.0190 g, 85%).

MS (APCI, ESI) m/z: 1403 (M+H)⁺.

Example 28: Drug-Linker 26

Step 1: Compound 28-1

The compound obtained in step 4 of Example 4 (1.12 g, 1.70 mmol) and4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)trifluoromethylbenzene(0.924 g, 3.40 mmol) were reacted in the same manner as in step 6 ofExample 3 to afford the desired compound (0.918 g, 83%).

MS (APCI, ESI) m/z: 657 [⁸¹Br, (M+H)⁺], 655 [⁷⁹Br, (M+H)⁺].

Step 2: Compound 28-2

The compound obtained in step 1 (0.918 g, 1.40 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.425 g, 60%).

MS (APCI, ESI) m/z: 511 [⁸¹Br, (M+H)⁺], 509 [⁷⁹Br, (M+H)⁺].

Step 3: Compound 28-3

The compound obtained in step 2 (0.425 g, 0.834 mmol) was reacted in thesame manner as in step 8 of Example 3. After a liquid separationoperation, the resultant was distillated under reduced pressure, and theresulting compound (0.410 g) was directly used for the subsequentreaction.

Step 4: Compound 28-4

The compound obtained in step 2 (0.425 g, 0.834 mmol) was reacted in thesame manner as in step 8 of Example 3 and step 9 of Example 3 to affordthe desired compound (0.420 g, 85%). MS (APCI, ESI) m/z: 597 [⁸¹Br,(M+H)⁺], 595 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 28-5

The compound obtained in step 4 (0.0960 g, 0.160 mmol) was reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.212 g, 99%).

MS (APCI, ESI) m/z: 1321 (M+H)⁺.

Step 6: Compound 28-6

The compound obtained in step 5 (0.210 g, 0.159 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.162 g, 84%).

MS (APCI, ESI) m/z: 1208 (M+H)⁺.

Step 7: Compound 28-7

The compound obtained in step 6 (0.160 g, 0.132 mmol) was used andreacted in the same manner as in step 12 of Example 3 to afford thedesired compound (0.103 g, 75%).

MS (APCI, ESI) m/z: 1039 (M+H)⁺.

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-5-oxo-2-[4-(trifluoromethyl)phenyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

Starting raw material 4-4 and the compound obtained in step 7 (0.101 g,0.0971 mmol) were reacted in the same manner as in step 13 of Example 3to afford the desired compound (0.107 g, 76%).

MS (APCI, ESI) m/z: 1441 (M+H)⁺.

Example 29: Drug-Linker 27

Step 1: tert-Buytl 1-[(prop-2en-1-yloxy)carbonyl]-L-prolyl-L-leucinate

tert-Butyl-L-prolyl-L-leucinate (4.64 g, 16.3 mmol) was reacted in thesame manner as in step 9 of Example 3, except that triethylamine (3.41mL, 24.5 mmol) was used in place of pyridine. After the completion ofthe reaction, a liquid separation operation was performed and theorganic solvent was distilled off under reduced pressure, and theresulting compound (5.79 g, 96%) was directly used for the subsequentreaction.

Step 2: 1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-L-leucine

To a solution of the compound obtained in step 1 (5.79 g, 15.7 mmol) indichloromethane (60 mL), trifluoroacetic acid (20 mL) was added, and theresultant was stirred at room temperature for 1 hour. Toluene was addedto the reaction solution, which was distillated under reduced pressure,and the resulting compound (2.69 g, 55%) was directly used for thesubsequent reaction.

Step 3:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-[4-(hydroxymethyl)phenyl]-L-leucinamide

To a solution of the compound obtained in step 2 (6.10 g, 19.5 mmol) inTHF (100 mL), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (5.07 g,20.5 mmol) was added, and the resultant was stirred at room temperaturefor 1 hour. Thereto, 4-aminobenzyl alcohol (2.16 g, 20.5 mmol) wasadded, and the resultant was stirred overnight. The reaction solutionwas distillated under reduced pressure, and the resulting residue wasdissolved in ethyl acetate. The resultant was washed with dilutehydrochloric acid, a saturated aqueous sodium hydrogen carbonate, andbrine, and the organic layer was dried over anhydrous sodium sulfate.After filtration, the solvent was distilled off under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=90:10 (v/v) to 0:100 (v/v)] toafford the desired compound (1.60 g, 20%).

¹H-NMR (CDCl₃) δ: 8.62-8.33 (1H, m), 7.68-7.49 (2H, m), 7.31-7.30 (2H,m), 6.66-6.43 (1H, m), 5.97-5.70 (1H, m), 5.39-4.96 (2H, m), 4.64-4.53(5H, m), 4.40-4.35 (1H, m), 3.57-3.50 (2H, m), 2.20-2.19 (2H, m),1.98-1.97 (3H, m), 1.66-1.64 (3H, m), 0.97-0.94 (6H, m).

MS (APCI, ESI) m/z: 418 (M+H)⁺.

Step 4:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-[4-({[(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamoyl]oxy}methyl)phenyl]-L-leucinamide

The compound obtained in step 3 (0.838 g, 2.01 mmol) was reacted in thesame manner as in step 6 of Example 1 to afford the desired compound(0.535 g, 37%).

¹H-NMR (CDCl₃) δ: 8.98 (1H, m), 8.62 (1H, s), 7.80 (1H, s), 7.65-7.50(2H, m), 7.33-7.31 (2H, m), 6.76 (1H, s), 6.67-6.34 (1H, m), 5.95-5.92(1H, m), 5.33-5.25 (1H, m), 5.30-5.27 (1H, m), 5.12 (2H, s), 4.66-4.63(3H, m), 4.58-4.55 (3H, m), 4.38-4.36 (1H, m), 3.99-3.96 (1H, m), 3.73(3H, s), 3.70-3.65 (2H, m), 3.57-3.55 (2H, m), 3.04-3.02 (1H, m),2.21-2.20 (3H, m), 1.98-1.95 (3H, m), 1.74-1.62 (3H, m), 1.32-1.30 (3H,m), 1.11-1.09 (16H, m), 0.97-0.94 (6H, m), 0.90 (9H, s), 0.60-0.52 (4H,m), 0.05-0.04 (6H, m).

Steps 5 and 6:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 4 (0.535 g, 0.531 mmol) was reacted in thesame manner as in step 7 of Example 1 and step 3 of Example 9 to affordthe desired compound (0.367 g, 78%).

¹H-NMR (CDCl₃) δ: 8.60 (1H, s), 7.58-7.42 (2H, m), 7.19-7.16 (3H, m),6.69-6.64 (2H, m), 5.89-5.87 (2H, m), 5.32-5.24 (2H, m), 5.10 (1H, m),4.93 (1H, m), 4.65-4.63 (1H, m), 4.57-4.54 (2H, m), 4.37-4.34 (1H, m),3.84 (3H, s), 3.72 (1H, m), 3.57-3.55 (3H, m), 3.40-3.38 (1H, m), 3.31(1H, m), 2.41 (1H, m), 2.20-2.18 (2H, m), 1.98-1.95 (3H, m), 1.73 (1H,m), 1.66-1.63 (1H, m), 1.16-1.10 (4H, m), 1.05-0.99 (18H, m) 0.97-0.93(6H, m), 0.71-0.64 (4H, m).

MS (APCI, ESI) m/z: 890 (M+H)⁺.

Step 7:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 6 (0.367 g, 0.412 mmol) was used andreacted in the same manner as in step 9 of Example 1 to afford thedesired compound (0.181 g, 44%).

¹H-NMR (CDCl₃) δ: 8.54 (1H, s), 7.52-7.45 (2H, m), 7.19 (1H, s), 7.14(2H, m), 6.71-6.68 (1H, m), 6.61 (1H, s), 6.01 (1H, m), 5.94-5.92 (1H,m), 5.34-5.17 (2H, m), 4.77 (1H, m), 4.64-4.56 (3H, m), 4.38-4.35 (1H,m), 3.85 (3H, s), 3.72-3.69 (1H, m), 3.55-3.46 (3H, m), 3.27 (1H, m),2.35 (1H, m), 2.21-2.18 (2H, m), 1.97-1.95 (3H, m), 1.54-1.51 (2H, m),1.16-1.09 (5H, m), 1.02-1.01 (18H, m), 0.97-0.93 (6H, m), 0.81 (9H, s),0.68-0.63 (4H, m), 0.19 (3H, s), 0.09 (3H, s).

Step 8:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 7 (0.181 g, 0.180 mmol) was treated in thesame manner as in step 10 of Example 1 to afford the desired compound(0.153 g, quantitative).

¹H-NMR (CDCl₃) δ: 8.57-8.27 (1H, m), 7.69-7.41 (2H, m), 7.23 (1H, s),7.12 (2H, m), 6.69-6.66 (1H, m), 6.64 (1H, s), 5.99-5.92 (3H, m),5.34-5.19 (3H, m), 4.81 (1H, m), 4.63-4.57 (4H, m), 4.38-4.36 (1H, m),3.94 (3H, s), 3.71 (1H, m), 3.54-3.52 (3H, m), 3.27 (1H, m), 2.35 (1H,m), 2.19 (2H, m), 1.97-1.95 (3H, m), 1.55-1.52 (2H, m), 0.97-0.93 (6H,m), 0.81 (9H, s), 0.76-0.61 (4H, m), 0.21 (3H, s), 0.09 (3H, s).

MS (APCI, ESI) m/z: 848 (M+H)⁺.

Step 9:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 8 (0.153 g, 0.180 mmol) was reacted in thesame manner as in step 9 of Example 4 to afford the desired compound(0.137 g, 57%).

¹H-NMR (CDCl₃) δ: 8.79 (1H, s), 7.54-7.51 (1H, m), 7.39-7.37 (2H, m),7.30-7.29 (2H, m), 7.23-7.21 (3H, m), 7.13-7.11 (2H, m), 6.88 (2H, m),6.82-6.80 (1H, m), 6.50-6.48 (1H, m), 6.03-6.01 (1H, m), 5.92-5.89 (1H,m), 5.75-5.72 (1H, m), 5.28-5.26 (3H, m), 5.06-5.03 (2H, m), 4.74-4.71(1H, m), 4.62-4.60 (4H, m), 4.36-4.34 (2H, m), 4.22-4.19 (3H, m),4.01-3.99 (1H, m), 3.88 (3H, s), 3.84-3.81 (6H, m), 3.71 (2H, m),3.53-3.50 (4H, m), 3.28-3.25 (2H, m), 2.72-2.68 (1H, m), 2.37-2.34 (4H,m), 2.19-2.16 (3H, m), 1.97-1.94 (2H, m), 1.49-1.43 (2H, m), 0.95-0.92(7H, m), 0.81 (9H, s), 0.68-0.65 (4H, m), 0.19 (3H, s), 0.13 (3H, s).

MS (APCI, ESI) m/z: 1324 (M+H)⁺.

Step 10:1-[(Prop-2-en-1-yloxy)carbonyl]-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 9 (0.136 g, 0.103 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.116 g, 93%).

¹H-NMR (CDCl₃) δ: 8.83 (1H, s), 7.57-7.52 (1H, m), 7.39-7.37 (2H, m),7.30-7.29 (2H, m), 7.24-7.20 (5H, m), 6.88 (2H, m), 6.81 (1H, s),6.54-6.51 (1H, m), 5.90-5.88 (2H, m), 5.76-5.74 (1H, m), 5.55-5.53 (1H,m), 5.33-5.05 (3H, m), 4.79-4.76 (1H, m), 4.63-4.60 (4H, m), 4.35-4.33(2H, m), 4.22-4.19 (3H, m), 4.04-4.03 (1H, m), 3.88 (3H, s), 3.85-3.83(6H, m), 3.72 (2H, m), 3.62-3.56 (4H, m), 3.32-3.29 (2H, m), 2.70 (1H,m), 2.45-2.38 (2H, m), 2.30-2.27 (2H, m), 2.20-2.10 (3H, m), 1.94-1.89(4H, m), 1.75-1.71 (2H, m), 0.95 (7H, m), 0.72-0.66 (4H, m).

MS (APCI, ESI) m/z: 1210 (M+H)⁺.

Step 11:L-Prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

The compound obtained in step 10 (0.116 g, 0.0958 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0530 g, 53%).

¹H-NMR (CDCl₃) δ: 9.15 (1H, s), 8.11 (1H, m), 7.50-7.45 (4H, m),7.32-7.31 (2H, m), 7.21 (1H, s), 7.13 (2H, m), 6.89 (2H, m), 6.37 (1H,s), 6.08 (1H, s), 5.88 (1H, m), 5.38 (1H, m), 4.69 (1H, m), 4.63-4.60(1H, m), 4.53-4.51 (1H, m), 4.31-4.28 (1H, m), 4.13-4.08 (3H, m), 3.89(3H, s), 3.83 (3H, s), 3.81 (3H, s), 3.75-3.70 (4H, m), 3.57-3.55 (3H,m), 3.37-3.33 (2H, m), 3.01-2.99 (1H, m), 2.90-2.86 (1H, m), 2.76-2.73(1H, m), 2.41 (1H, m), 2.17-2.15 (3H, m), 1.90-1.87 (1H, m), 1.74 (4H,m), 1.25 (2H, m), 0.97-0.91 (7H, m), 0.67 (4H, m).

MS (APCI, ESI) m/z: 1042 (M+H)⁺.

Step 12:N-[(9H-Fluoren-9-yloxy)carbonyl]glycylglycyl-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

To a solution of the compound obtained in step 11 (0.0410 g, 0.0393mmol) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycine (0.0410 g,0.0393 mmol) in N,N-dimethylformamide (1 mL), 1-hydroxybenzotriazolemonohydrate (0.000602 g, 0.00393 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.00905 g,0.0472 mmol) were added, and the resultant was stirred at roomtemperature for 1 hour. The resultant was distillated under reducedpressure, and the resulting residue was purified by silica gel columnchromatography (chloroform:methanol=100:0 to 90:10) to afford thedesired compound (0.0520 g, 94%).

¹H-NMR (CDCl₃) δ: 8.57 (1H, s), 7.77-7.75 (3H, m), 7.63-7.61 (2H, m),7.51-7.48 (2H, m), 7.40-7.38 (3H, m), 7.31-7.29 (4H, m), 7.21-7.11 (5H,m), 6.88 (2H, m), 6.47 (1H, s), 6.08 (1H, s), 5.93 (1H, m), 5.21-5.19(1H, m), 5.13 (1H, m), 4.78 (1H, m), 4.66-4.59 (1H, m), 4.53-4.51 (1H,m), 4.42-4.39 (1H, m), 4.33-4.31 (1H, m), 4.19-4.11 (6H, m), 4.10-3.86(4H, m), 3.82 (3H, s), 3.76 (3H, s), 3.73-3.71 (3H, m), 3.56-3.51 (7H,m), 3.32-3.29 (2H, m), 2.73-2.69 (1H, m), 2.40 (1H, m), 2.29-2.27 (3H,m), 2.06-2.04 (4H, m), 1.73-1.70 (2H, m), 1.25 (2H, m), 0.96-0.93 (6H,m), 0.66 (4H, m).

MS (APCI, ESI) m/z: 1378 (M+H)⁺.

Step 13:Glycylglycyl-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

To a solution of the compound obtained in step 12 (0.0520 g, 0.0377mmol) in N,N-dimethylformamide (2 mL),1,8-diazabicyclo[5.4.0]-7-undecene (67 μL, 0.0453 mmol) was added, andthe resultant was stirred at room temperature for 1 hour. The resultantwas distillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [chloroform:CMW=100:0 (v/v)to 0:100 (v/v)] to afford the desired compound (0.0420 g, 92%).

¹H-NMR (CDCl₃) δ: 8.64 (1H, s), 7.74 (1H, m), 7.66-7.64 (1H, m), 7.50(1H, s), 7.45 (1H, s), 7.33-7.30 (3H, m), 7.24-7.21 (2H, m), 7.18-7.16(3H, m), 6.89 (2H, m), 6.51 (1H, s), 6.15 (1H, s), 5.89 (1H, m), 5.27(1H, m), 4.82-4.78 (1H, m), 4.74 (1H, m), 4.56-4.54 (1H, m), 4.49-4.47(1H, m), 4.23-4.20 (3H, m), 4.13-4.10 (3H, m), 3.96-3.92 (1H, m), 3.90(3H, s), 3.83 (3H, s), 3.76 (3H, s), 3.72 (2H, m), 3.62-3.56 (5H, m),3.40-3.36 (1H, m), 3.31 (1H, m), 3.04 (1H, m), 2.90-2.86 (1H, m),2.75-2.71 (1H, m), 2.42-2.39 (1H, m), 2.36 (2H, s), 2.29-2.27 (3H, m),2.07-2.05 (3H, m), 1.97-1.95 (1H, m), 1.75-1.72 (2H, m), 0.94 (6H, m),0.69 (4H, m).

MS (APCI, ESI) m/z: 1156 (M+H)⁺.

Step 14: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-prolyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-leucinamide

To a solution of the compound obtained in step 13 (0.0400 g, 0.0346mmol) in N,N-dimethylformamide (1 mL),1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidin-2,5-dione (0.0140 g, 0.0346 mmol,Click Chemistry Tools) and diisopropylamine (0.0240 mL, 0.138 mmol) wereadded, and the resultant was stirred at room temperature for 18 hours.The reaction solution was distillated under reduced pressure, and theresulting residue was purified by silica gel column chromatography[chloroform:methanol=100:0 (v/v) to 90:10 (v/v)] to afford the desiredcompound (0.0230 g, 46%).

MS (APCI, ESI) m/z: 1443 (M+H)⁺.

Example 30: Drug-Linker 28

Step 1:N-{[(1R,8S)-Bicyclo[6.1.0]non-4-in-9-ylmethoxy]carbonyl}glycylglycine

To a solution of starting material 30-1 (0.215 g, 0.682 mmol,Chemistry-A European Journal 2016, 22, 639) in N,N-dimethylformamide (4mL), N,N-diisopropylethylamine (0.119 mL, 0.682 mmol), glycyl-glycine(0.0900 g, 0.682 mmol), and water (2 mL) were added, and the resultantwas stirred at room temperature overnight. The reaction solution wasdistillated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography [chloroform:CMW=100:0 (v/v)to 0:100 (v/v)] to afford the desired compound (0.205 g, 98%).

MS (APCI, ESI) m/z: 309 (M+H)⁺.

Step 2:N-{[(1R,8S)-Bicyclo[6.1.0]non-4-in-9-ylmethoxy]carbonyl}glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 1 (0.0180 g, 0.0587 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0420 g, 60%).

¹H-NMR (DMSO-D₆) δ: 9.93 (1H, s), 8.25 (1H, m), 8.05 (1H, s), 7.85 (1H,m), 7.60-7.54 (2H, m), 7.45 (1H, s), 7.39-7.37 (3H, m), 7.29 (1H, s),7.20 (1H, m), 7.04 (1H, s), 6.91 (2H, m), 6.72 (1H, s), 6.56-6.53 (2H,m), 6.31 (1H, s), 5.76-5.74 (1H, m), 5.19 (1H, m), 4.80 (1H, m),4.38-4.36 (1H, m), 4.23-4.21 (2H, m), 4.04 (2H, m), 3.95-3.92 (3H, m),3.78-3.76 (9H, m), 3.66 (3H, s), 3.60 (2H, m), 3.55-3.52 (2H, m),3.45-3.38 (2H, m), 3.26-3.23 (1H, m), 3.14 (1H, m), 2.77-2.74 (1H, m),2.35-2.33 (1H, m), 2.20-2.11 (6H, m), 1.99-1.96 (1H, m), 1.81-1.78 (4H,m), 1.56-1.54 (5H, m), 1.29-1.26 (4H, m), 0.87-0.82 (9H, m), 0.67-0.62(4H, m).

MS (APCI, ESI) m/z: 1320 (M+H)⁺.

Example 31: Drug-Linker 29

Step 1: Compound 31-1

The compound obtained in step 4 of Example 4 (1.00 g, 1.52 mmol) and4-methylphenylboronic acid (0.309 g, 2.27 mmol) were reacted in the samemanner as in step 6 of Example 3 to afford the desired compound (0.653g, 72%).

Step 2: Compound 31-2

The compound obtained in step 1 (0.653 g, 1.09 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.377 g, 76%).

MS (APCI, ESI) m/z: 457 [⁸¹Br, (M+H)⁺], 455 [⁷⁹Br, (M+H)⁺].

Step 3: Compound 31-3

The compound obtained in step 2 (0.377 g, 0.828 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.311 g, 82%).

MS (APCI, ESI) m/z: 459 [⁸¹Br, (M+H)⁺], 457 [⁷⁹Br, (M+H)⁺].

Step 4: Compound 31-4

The compound obtained in step 3 (0.311 g, 0.68 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.320 g, 87%).

MS (APCI, ESI) m/z: 543 [⁸¹Br, (M+H)⁺], 541 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 31-5

The compound obtained in step 4 (0.0737 g, 0.136 mmol) was reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.145 g, 92%).

MS (APCI, ESI) m/z: 1272 (M+H)⁺.

Step 6: Compound 31-6

The compound obtained in step 5 (0.145 g, 0.114 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.122 g, 93%).

MS (APCI, ESI) m/z: 1158 (M+H)⁺.

Step 7: Compound 31-7

The compound obtained in step 6 (0.122 g, 0.114 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.0598 g, 53%).

MS (APCI, ESI) m/z: 990 (M+H)⁺.

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.0300 g, 0.0304 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0184 g, 44%).

MS (APCI, ESI) m/z: 1391 (M+H)⁺.

Example 32: Drug-Linker 30

Step 1: Compound 32-1

The compound obtained in step 4 of Example 4 (1.00 g, 1.52 mmol) and4-fluorophenylboronic acid (0.318 g, 2.27 mmol) were reacted in the samemanner as in step 6 of Example 3 to afford the desired compound (0.623g, 68%).

Step 2: Compound 32-2

The compound obtained in step 1 (0.623 g, 1.03 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.244 g, 52%).

MS (APCI, ESI) m/z: 461 [⁸¹Br, (M+H)⁺], 459 [⁷⁹Br, (M+H)⁺].

Step 3: Compound 32-3

The compound obtained in step 2 (0.244 g, 0.531 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.144 g, 59%).

MS (APCI, ESI) m/z: 463 [⁸¹Br, (M+H)⁺], 461 [⁷⁹Br, (M+H)⁺].

Step 4: Compound 32-4

The compound obtained in step 3 (0.144 g, 0.312 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.139 g, 82%).

MS (APCI, ESI) m/z: 547 [⁸¹Br, (M+H)⁺], 545 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 32-5

The compound obtained in step 4 (0.0742 g, 0.136 mmol) was reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.138 g, 88%).

MS (APCI, ESI) m/z: 1284 (M+H)⁺.

Step 6: Compound 32-6

The compound obtained in step 5 (0.138 g, 0.108 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.109 g, 87%).

MS (APCI, ESI) m/z: 1170 (M+H)⁺.

Step 7: Compound 32-7

The compound obtained in step 6 (0.109 g, 0.101 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.0613 g, 61%).

MS (APCI, ESI) m/z: 1002 (M+H)⁺.

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-8′-(3-{[(11aS)-2-(4-fluorophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-11′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.0300 g, 0.0333 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0191 g, 45%).

MS (APCI, ESI) m/z: 1402 (M+H)⁺.

Example 33: Drug-Linker 31

Step 1: Compound 33-1

The compound obtained in step 4 of Example 4 (1.00 g, 1.52 mmol) andthiophene-3-boronic acid (0.582 g, 4.55 mmol) were used and reacted inthe same manner as in step 6 of Example 3 to afford the desired compound(0.611 g, 68%).

Step 2: Compound 33-2

The compound obtained in step 1 (0.611 g, 1.03 mmol) was used andreacted in the same manner as in step 7 of Example 3 to afford thedesired compound (0.397 g, 86%).

MS (APCI, ESI) m/z: 449 [⁸¹Br, (M+H)⁺], 447 [⁷⁹Br, (M+H)⁺].

Step 3: Compound 33-3

The compound obtained in step 2 (0.397 g, 0.887 mmol) was used andreacted in the same manner as in step 8 of Example 3 to afford thedesired compound (0.341 g, 86%).

MS (APCI, ESI) m/z: 451 [⁸¹Br, (M+H)⁺], 449 [⁷⁹Br, (M+H)⁺].

Step 4: Compound 33-4

The compound obtained in step 3 (0.341 g, 0.759 mmol) was used andreacted in the same manner as in step 9 of Example 3 to afford thedesired compound (0.368 g, 91%).

MS (APCI, ESI) m/z: 535 [⁸¹Br, (M+H)⁺], 533 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 33-5

The compound obtained in step 4 (0.0726 g, 0.136 mmol) was used andreacted in the same manner as in step 10 of Example 3 to afford thedesired compound (0.125 g, 80%).

MS (APCI, ESI) m/z: 1260 (M+H)⁺.

Step 6: Compound 33-6

The compound obtained in step 5 (0.125 g, 0.0992 mmol) was used andreacted in the same manner as in step 11 of Example 3 to afford thedesired compound (0.109 g, 96%).

MS (APCI, ESI) m/z: 1146 (M+H)⁺.

Step 7: Compound 33-7

The compound obtained in step 6 (0.109 g, 0.0951 mmol) was used andreacted in the same manner as in step 12 of Example 3 to afford thedesired compound (0.0723 g, 78%).

¹H-NMR (CDCl₃) δ: 9.13-9.04 (1H, m), 7.89-7.87 (1H, m), 7.47-7.42 (4H,m), 7.27-7.23 (3H, m), 7.18 (1H, s), 7.12-7.08 (2H, m), 6.97-6.96 (1H,m), 6.42-6.37 (1H, m), 6.08-6.04 (1H, m), 5.86-5.84 (1H, m), 5.34-5.31(1H, m), 4.65-4.58 (3H, m), 4.23-3.95 (5H, m), 3.85 (3H, s), 3.75-3.69(6H, m), 3.57-3.47 (3H, m), 3.34-3.29 (3H, m), 2.72-2.68 (1H, m),2.38-2.29 (2H, m), 2.15-2.14 (2H, m), 1.72-1.69 (1H, m), 1.40-1.38 (3H,m), 0.96-0.95 (3H, m), 0.80-0.61 (7H, m).

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(aS)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.0300 g, 0.0307 mmol) was used andreacted in the same manner as in step 13 of Example 3 to afford thedesired compound (0.0101 g, 24%).

MS (APCI, ESI) m/z: 1379 (M+H)⁺.

Example 34: Drug-Linker 32

Step 1: Compound 34-2

To a solution of starting material 34-1 (4.00 g, 10.8 mmol, US20150283262) in dichloromethane (100 mL), N,N-dimethylformamide (0.2 mL)and oxalyl chloride (2.75 g, 21.7 mmol) were added at 0° C., a followedby stirring at room temperature for 30 minutes. After the solvent wasdistilled off under reduced pressure, the residue was dried underreduced pressure, and dissolved in dichloromethane (60 mL). Thereto,(2S)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2,3-dihydro-1H-indole(4.28 g, 16.2 mmol, Journal of the American Chemical Society 2006, 128,14264) and triethylamine (1.64 g, 16.2 mmol) were added at 0° C.,followed by stirring at room temperature for 15 minutes. Water was addedto the reaction solution, which was extracted with dichloromethane. Theorganic layer obtained was washed with brine, and dried over magnesiumsulfate, and then distillated under reduced pressure. The resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=100:0 (v/v) to 80:20 (v/v)] to afford the desired compound (6.10g, 92%).

MS (APCI, ESI) m/z: 615 (M+H)⁺

Step 2: Compound 34-3

To a solution of the compound obtained in step 1 (6.10 g, 9.90 mmol) inethanol (100 mL), 5% palladium carbon (moisture content: 54%, 1.00 g)was added under the nitrogen atmosphere, and the reaction solution wasthen stirred under the hydrogen atmosphere at room temperature for 4hours. After the reaction solution was filtered through a Celite, thefiltrate was distillated under reduced pressure to afford the desiredcompound (5.80 g, quantitative).

MS (APCI, ESI) m/z: 585 (M+H)⁺

Step 3: Compound 34-4

The compound obtained in step 2 (2.90 g, 5.00 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(3.20 g, 96%).

¹H-NMR (CDCl₃) δ: 8.26 (1H, s), 7.78 (1H, s), 7.18-7.17 (1H, m),6.94-6.83 (3H, m), 5.94-5.90 (1H, m), 5.35-5.19 (2H, m), 4.74 (1H, m),4.65-4.60 (2H, m), 3.76-3.61 (6H, m), 3.31-3.27 (1H, m), 3.10 (1H, m),1.35-1.17 (3H, m), 1.10 (18H, m), 0.79-0.70 (9H, m),−0.03 (3H, s),−0.08(3H, s).

Step 4: Compound 34-5

The compound obtained in step 3 (3.20 g, 4.80 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(2.32 g, 87%).

MS (APCI, ESI) m/z: 555 (M+H)⁺

Step 5: Compound 34-6

The compound obtained in step 4 (2.32 g, 4.18 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(1.68 g, 73%).

MS (APCI, ESI) m/z: 553 (M+H)⁺

Step 6: Compound 34-7

The compound obtained in step 5 (1.68 g, 3.04 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(2.32 g, quantitative).

¹H-NMR (CDCl₃) δ: 8.16 (1H, m), 7.28-7.20 (3H, m), 7.09-7.07 (1H, m),6.70 (1H, s), 5.80-5.76 (2H, m), 5.14-5.12 (2H, m), 4.60 (1H, m), 4.37(1H, m), 4.01-3.99 (1H, m), 3.87 (3H, s), 3.45-3.41 (1H, m), 2.99-2.95(1H, m), 1.28-1.23 (3H, m), 1.10-1.07 (18H, m), 0.92 (9H, s), 0.26 (3H,s), 0.19 (3H, s).

Step 7: Compound 34-8

The compound obtained in step 6 (2.32 g, 3.48 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(1.42 g, 80%).

MS (APCI, ESI) m/z: 511 (M+H)⁺

Step 8: Compound 34-9

The compound obtained in step 7 (0.720 g, 1.41 mmol) was reacted in thesame manner as in step 1 of Example 4 to afford the desired compound(0.580 g, 65%).

MS (APCI, ESI) m/z: 633 [⁸¹Br, (M+H)⁺], 631 [⁷⁹Br, (M+H)⁺].

Step 9: Compound 34-10

The compound obtained in step 8 (0.235 g, 0.371 mmol) was reacted in thesame manner as in step 3 of Example 10 to afford the desired compound(0.347 g, 83%).

MS (APCI, ESI) m/z: 1359 (M+H)⁺

Step 10: Compound 34-11

The compound obtained in step 9 (0.347 g, 0.255 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford a silyl-deprotectedform (0.265 g, 92%). The silyl-deprotected form (0.265 g, 0.234 mmol)was reacted in the same manner as in step 12 of Example 1 to afford thedesired compound (0.086 g, 39%).

MS (APCI, ESI) m/z: 944 (M+H)⁺

Step 11: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(5aS)-10-methoxy-12-oxo-5a,12-dihydro-5H-indolo[2,1-c][1,4]benzodiazepin-9-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 10 (0.0860 g, 0.0911 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0800 g, 65%).

MS (APCI, ESI) m/z: 1345 (M+H)⁺

Example 35: Drug-Linker 33

Step 1: Compound 35-1

The compound obtained in step 7 of Example 34 (0.700 g, 1.37 mmol) wasreacted in the same manner as in step 2 of Example 3 to afford thedesired compound (0.790 g, 87%).

MS (APCI, ESI) m/z: 661 [⁸¹Br, (M+H)⁺], 659 [⁷⁹Br, (M+H)⁺].

Step 2: Compound 35-2

The compound obtained in step 1 (0.245 g, 0.371 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.368 g, 86%).

MS (APCI, ESI) m/z: 1387 (M+H)⁺

Step 3: Compound 35-3

The compound obtained in step 2 (0.368 g, 0.265 mmol) was reacted in thesame manner as in steps 11 and 12 of Example 3 to afford the desiredcompound (0.180 g, 81%).

MS (APCI, ESI) m/z: 972 (M+H)⁺

Step 4: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(5aS)-10-methoxy-12-oxo-5a,12-dihydro-5H-indolo[2,1-c][1,4]benzodiazepin-9-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 3 (0.0700 g, 0.0720 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0440 g, 44%).

MS (APCI, ESI) m/z: 1373 (M+H)⁺

Example 36: Drug-Linker 34

Step 1:(11aS)-8-[(5-Bromopentyl)oxy]-7-methoxy-5,11-dioxo-10-{[2-(trimethylsilyl)ethoxy]methyl}-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-carbaldehyde

The compound obtained in step 5 of Example 3 (3.54 g, 5.15 mmol) wasdissolved in N,N-dimethylformamide (52 mL), to which N-formylsaccharin(5.44 g, 25.7 mmol), palladium acetate (0.0578 g, 0.257 mmol),1,4-bis(diphenylphosphino)butane (0.154 g, 0.360 mmol), sodium carbonate(2.73 g, 25.7 mmol), and triethylsilane (1.20 g, 10.3 mmol) were added,and the resultant was stirred at room temperature for 19 hours. Waterwas added to the reaction solution, and the reaction solution wasextracted with ethyl acetate, and the extract was washed with water andbrine. The organic layer was dried over magnesium sulfate, and thendistillated under reduced pressure. The resulting residue was purifiedby silica gel column chromatography [hexane:ethyl acetate=100:0 (v/v) to40:60 (v/v)] to afford the desired compound (0.760 g, 26%).

¹H-NMR (CDCl₃) δ: 9.74 (1H, s), 7.79 (1H, s), 7.32 (1H, s), 7.24 (1H,s), 5.54 (1H, m), 4.76-4.69 (2H, m), 4.07-4.05 (2H, m), 3.93 (3H, s),3.83-3.65 (3H, m), 3.45 (2H, m), 3.02-2.98 (1H, m), 1.95-1.91 (4H, m),1.69-1.58 (2H, m), 1.02-0.92 (2H, m), 0.06-0.03 (9H, m).

Step 2:(11aS)-8-[(5-Bromopentyl)oxy]-2-(hydroxymethyl)-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

The compound obtained in step 1 (0.760 g, 1.34 mmol) was dissolved indichloromethane (14 mL), to which sodium borohydride (0.101 g, 2.68mmol) was added at −78° C., and the temperature was then raised to roomtemperature. To the reaction solution, 1 N hydrochloric acid was added,and the organic layer was washed with water and brine. The organic layerwas dried over magnesium sulfate, and then distillated under reducedpressure. The resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)] toafford the desired compound (0.432 g, 57%).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.23 (1H, s), 6.93 (1H, s), 5.53 (1H,m), 4.67 (1H, m), 4.57-4.54 (1H, m), 4.32-4.31 (2H, m), 4.10-4.01 (2H,m), 3.92 (3H, s), 3.83-3.57 (3H, m), 3.45 (2H, m), 2.90-2.88 (1H, m),1.99-1.88 (4H, m), 1.69-1.61 (2H, m), 1.00-0.98 (2H, m), 0.03 (9H, s).

MS (APCI, ESI) m/z: 571 [⁸¹Br, (M+H)⁺], 569 [⁷⁹Br, (M+H)⁺].

Step 3:(11aS)-8-[(5-Bromopentyl)oxy]-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-methoxy-10-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

The compound obtained in step 2 (0.432 g, 0.758 mmol) was dissolved inN,N-dimethylformamide (8 mL), to which imidazole (0.0775 g, 1.14 mmol)and tert-butyldimethylsilyl chloride (0.137 g, 0.910 mmol) were added atroom temperature, and the resultant was stirred at room temperature for10 minutes. Water was added to the reaction solution, and the reactionsolution was extracted with ethyl acetate. The organic layer was washedwith water and brine, and dried over magnesium sulfate. Afterdistillation under reduced pressure, the resulting residue was purifiedby silica gel column chromatography [hexane:ethyl acetate=100:0 (v/v) to30:70 (v/v)] to afford the desired compound (0.485 g, 94%).

¹H-NMR (CDCl₃) δ: 7.35 (1H, s), 7.22 (1H, s), 6.87 (1H, m), 5.53 (1H,m), 4.68 (1H, m), 4.54-4.52 (1H, m), 4.33-4.28 (2H, m), 4.11-4.00 (2H,m), 3.92 (3H, s), 3.80-3.78 (1H, m), 3.69-3.66 (1H, m), 3.52-3.50 (1H,m), 3.45 (2H, m), 2.86-2.82 (1H, m), 1.99-1.87 (4H, m), 1.68-1.60 (2H,m), 1.00-0.98 (2H, m), 0.91 (9H, s), 0.09 (6H, m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 685 [⁸¹Br, (M+H)⁺], 683 [⁷⁹Br, (M+H)⁺].

Step 4:(11aS)-8-[(5-Bromopentyl)oxy]-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-methoxy-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 3 (0.103 g, 0.151 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.0590 g, 73%).

¹H-NMR (CDCl₃) δ: 7.83 (1H, m), 7.51 (1H, s), 6.92 (1H, m), 6.79 (1H,s), 4.32-4.30 (3H, m), 4.12-4.04 (2H, m), 3.93 (3H, s), 3.86-3.84 (1H,m), 3.46-3.39 (2H, m), 3.26-3.23 (1H, m), 3.07-3.03 (1H, m), 1.97-1.85(4H, m), 1.68-1.63 (2H, m), 0.92 (9H, s), 0.09 (6H, s).

MS (APCI, ESI) m/z: 539 [⁸¹Br, (M+H)⁺], 537 [⁷⁹Br, (M+H)⁺].

Step 5:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-[(5-{[(11aS)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 4 (0.251 g, 0.467 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.300 g, 51%).

Step 6:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-[(5-{[(11aS)-2-(hydroxymethyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 5 (0.300 g, 0.237 mmol) was reacted in thesame manner as in steps 11 and 12 of Example 33 to afford the desiredcompound (0.0540 g, 53%).

MS (APCI, ESI) m/z: 952 (M+H)⁺

Step 7: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-8′-[(5-{[(11aS)-2-(hydroxymethyl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 6 (0.0500 mg, 0.0525 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0340 mg, 48%).

MS (APCI, ESI) m/z: 1351 (M−H)⁻

Example 37: Drug-Linker 35

Step 1: Methyl(6S)-5-[4-(benzyloxy)-5-methoxy-2-nitrobenzoyl]-5-azaspiro[2.4]heptane-6-carboxylate

To a solution of 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (6.07 g,20.0 mmol, Tetrahedron 1995, 51, 5617) and N,N-dimethylformamide (1.08mL, 13.9 mmol) in dichloromethane (100 mL), oxalyl chloride (3.43 mL,40.0 mmol) was added dropwise under ice-cooling over 5 minutes. Thereaction solution was stirred at room temperature for 5 hours, and thendistillated under reduced pressure, and the resulting residue wasdissolved in dichloromethane (20 mL), which was distillated underreduced pressure. After this operation was repeated three times, theresidue was suspended in dichloromethane (5 mL), to which excessiveamounts of diethyl ether and hexane were added, and the followingfiltration and drying under reduced pressure afforded the crude acylchloride. The acyl chloride obtained was dissolved in dichloromethaneand cooled to −40° C. (dry ice-acetonitrile bath), to which methyl(6S)-5-azaspiro[2.4]heptane-6-carboxylate hydrochloride (4.22 g, 22.0mmol, Tetrahedron Letters 2012. 53. 3847) and triethylamine (3.36 mL,24.2 mmol) were gradually added. The temperature of the reaction mixturewas raised to room temperature overnight. To the reaction mixture, 1 Nhydrochloric acid was added, and the reaction mixture was extracted withdichloromethane. The organic layer was washed with water, a saturatedaqueous sodium hydrogen carbonate, and brine, and dried over anhydroussodium sulfate. The resultant was distillated under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 to 50:50] to afford thedesired compound (6.55 g, 80%).

MS (APCI, ESI) m/z: 441 (M+H)⁺

Step 2:(11a′S)-8′-(Benzyloxy)-7′-methoxy-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′,11′(10′H,11a′H)-dione

To a solution of the compound obtained in step 1 (6.55 g, 16.0 mmol) inethanol (150 mL) and THF (150 mL), Raney nickel (7.00 g) was added underthe nitrogen atmosphere. Hydrazine monohydrate (7 mL) was added to thereaction mixture, and the temperature was gradually raised to 50° C.After stirring at 50° C. for 2 hours, Raney nickel (3.00 g) andhydrazine monohydrate (3 mL) were added thereto, and the resultant wasstirred for 1 hour. THF (100 mL) was added to the reaction mixture,which was filtered through a Celite. The resultant was distillated underreduced pressure, and the resulting residue was purified by silica gelcolumn chromatography [hexane:ethyl acetate=100:0 (v/v) to 25:75 (v/v)]to afford the desired compound (4.42 g, 73%).

¹H-NMR (CDCl₃) δ: 7.82 (1H, s), 7.48 (1H, s), 7.42-7.35 (4H, m),7.32-7.31 (1H, m), 6.44 (1H, s), 5.16 (2H, s), 4.16-4.10 (1H, m), 3.93(3H, s), 3.78-3.76 (1H, m), 3.39-3.37 (1H, m), 2.45-2.43 (1H, m),2.24-2.21 (1H, m), 0.83-0.61 (4H, m).

MS (APCI, ESI) m/z: 379 (M+H)⁺

Step 3:(11a′S)-8′-(Benzyloxy)-7′-methoxy-10′-{[2-(trimethylsilyl)ethoxy]methyl}-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′,11′(10′H,11a′H)-dione

To a solution of the compound obtained in step 2 (10.0 g, 26.4 mmol) inTHF (150 mL), a 2.6 mol/L normal-hexane solution of normal-butyllithium(12.0 mL, 31.8 mmol) was added slowly dropwise at −40° C. The reactionsolution was stirred at −40° C. for 15 minutes, and2-(chloromethoxy)ethyltrimethylsilane (5.57 mL, 31.7 mmol) was thenadded slowly dropwise thereto. After the reaction solution was stirredat room temperature for 3 hours, water was added thereto, and theresultant was extracted with ethyl acetate. The organic layer was washedwith water and brine, and dried over anhydrous sodium sulfate. Afterdistillation under reduced pressure, the resulting residue was purifiedby silica gel column chromatography [hexane:ethyl acetate=100:0 (v/v) to30:70 (v/v)] to afford the desired compound (11.8 g, 88%).

¹H-NMR (CDCl₃) δ: 7.45-7.44 (2H, m), 7.37-7.32 (4H, m), 7.28 (1H, s),5.48-5.46 (1H, m), 5.21 (2H, s), 4.50-4.48 (1H, m), 4.22-4.20 (1H, m),3.95 (3H, s), 3.73-3.70 (2H, m), 3.62-3.60 (1H, m), 3.41-3.38 (1H, m),2.45-2.43 (1H, m), 2.23-2.20 (1H, m), 0.98-0.96 (2H, m), 0.83-0.68 (4H,m), 0.04 (9H, s).

MS (APCI, ESI) m/z: 509 (M+H)⁺

Step 4:(11a′S)-8′-Hydroxy-7′-methoxy-10′-{[2-(trimethylsilyl)ethoxy]methyl}-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′,11′(10′H,11a′H)-dione

To a solution of the compound obtained in step 3 (18.7 g, 36.8 mmol) inTHF (50 mL) and ethanol (100 mL), a 5% palladium carbon catalyst (5.00g) was added under the nitrogen atmosphere. The nitrogen balloon wasimmediately replaced with a hydrogen balloon, and the reaction mixturewas stirred under the hydrogen atmosphere for 6 hours. The reactionmixture was diluted by addition of chloroform and filtered through aCelite, and the filtrate was then distillated under reduced pressure,and the resulting residue was purified by silica gel columnchromatography [hexane:ethyl acetate=100:0 (v/v) to 25:75 (v/v)] toafford the desired compound (15.1 g, 98%).

¹H-NMR (CDCl₃) δ: 7.38 (1H, s), 7.28 (1H, s), 6.01 (1H, s), 5.49-5.47(1H, m), 4.70-4.68 (1H, m), 4.24-4.22 (1H, m), 3.96 (3H, s), 3.76-3.71(2H, m), 3.66-3.64 (1H, m), 3.42-3.39 (1H, m), 2.47-2.45 (1H, m),2.23-2.21 (1H, m), 1.01-0.99 (2H, m), 0.89-0.63 (4H, m), 0.03 (9H, s).

MS (APCI, ESI) m/z: 419 (M+H)⁺

Step 5:(11a′S)-8′-[(5-Bromopentyl)oxy]-7′-methoxy-10′-{[2-(trimethylsilyl)ethoxy]methyl}-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′,11′(10′H,11a′H)-dione

The compound obtained in step 4 (2.77 g, 6.62 mmol) was reacted in thesame manner as in step 2 of Example 3 to afford the desired compound(3.31 g, 88%).

¹H-NMR (CDCl₃) δ: 7.36 (1H, s), 7.25 (1H, s), 5.55 (1H, m), 4.65 (1H,m), 4.24-4.23 (1H, m), 4.11-4.03 (2H, m), 3.93 (3H, s), 3.85-3.78 (1H,m), 3.72-3.69 (2H, m), 3.46-3.39 (3H, m), 2.47-2.44 (1H, m), 2.25-2.22(1H, m), 1.95-1.91 (4H, m), 1.67-1.59 (1H, m), 1.03-0.95 (2H, m),0.90-0.85 (1H, m), 0.70-0.66 (4H, m), 0.05 (9H, s).

Step 6:(11a′S)-8′-[(5-Bromopentyl)oxy]-7′-methoxy-11′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 5 (3.31 g, 5.83 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(1.11 g, 45%).

¹H-NMR (CDCl₃) δ: 7.81 (1H, m), 7.53 (1H, s), 6.82 (1H, s), 4.13-4.06(2H, m), 3.97 (3H, s), 3.88-3.83 (1H, m), 3.69 (1H, m), 3.52-3.39 (3H,m), 2.55-2.52 (1H, m), 2.06-1.89 (5H, m), 1.67-1.63 (2H, m), 0.76-0.72(4H, m).

Step 7:(11a′S)-8′-[(5-Bromopentyl)oxy]-7′-methoxy-1′,10′,11′,11a′-tetrahydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 6 (2.56 g, 6.08 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(1.15 g, 45%).

¹H-NMR (CDCl₃) δ: 7.60 (1H, s), 6.07 (1H, s), 4.11-4.04 (1H, m), 3.99(2H, m), 3.87-3.84 (1H, m), 3.85 (3H, s), 3.73 (1H, m), 3.58-3.53 (2H,m), 3.47-3.42 (3H, m), 2.03-1.78 (6H, m), 1.65-1.63 (2H, m), 0.77-0.56(4H, m).

Step 8: Prop-2-en-1-yl(11a′S)-8′-[(5-bromopentyl)oxy]-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 7 (1.15 g, 2.72 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(1.14 g, 82%).

¹H-NMR (CDCl₃) δ: 7.23 (1H, s), 6.69 (1H, s), 5.79 (1H, s), 5.13-5.10(2H, m), 4.68-4.66 (1H, m), 4.48-4.45 (2H, m), 4.01 (2H, m), 3.92 (3H,s), 3.76 (1H, m), 3.54-3.37 (3H, m), 2.39 (1H, m), 1.95-1.90 (4H, m),1.68-1.61 (3H, m), 1.44 (1H, m), 0.75-0.66 (4H, m).

Step 9:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-{[5-({(11a′S)-7′-methoxy-5′-oxo-10′-[(prop-2-en-1-yloxy)carbonyl]-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 8 (0.374 g, 0.737 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.589 g, 65%).

MS (APCI, ESI) m/z: 1234 (M+H)⁺

Step 10:N-[(Prop-2-en-1-yloxy)carbonyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-{[5-({(11a′S)-7′-methoxy-5′-oxo-10′-[(prop-2-en-1-yloxy)carbonyl]-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 5 (0.589 g, 0.477 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.382 g, 71%).

¹H-NMR (CDCl₃) δ: 8.90 (1H, s), 7.55 (2H, m), 7.25-7.21 (2H, m), 6.74(2H, m), 6.38 (1H, s), 5.90-5.87 (5H, m), 5.33-5.09 (8H, m), 4.66-4.60(8H, m), 3.98-3.91 (10H, m), 3.77-3.30 (12H, m), 2.42-2.36 (2H, m),1.77-1.39 (6H, m), 0.91-0.70 (14H, m).

Step 11:L-Valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 10 (0.382 g, 0.341 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.200 g, 62%).

MS (APCI, ESI) m/z: 952 (M+H)⁺

Step 12: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 11 (0.0560 g, 0.0588 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.0500 g, 63%).

MS (APCI, ESI) m/z: 1354 (M+H)⁺

Example 38: Drug-Linker 36

Step 1: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 11 of Example 37 (0.0410 g, 0.0430 mmol)was reacted in the same manner as in step 1 of Example 6 to afford thedesired compound (0.0210 g, 39%).

MS (APCI, ESI) m/z: 1240 (M+H)⁺

Example 39: Drug-Linker 37

Step 1: Compound 39-1

The compound obtained in step 4 of Example 4 (1.00 g, 1.52 mmol) andphenylboronic acid (0.370 g, 3.03 mmol) were used and reacted in thesame manner as in step 6 of Example 3 to afford the desired compound(0.726 g, 81%).

MS (APCI, ESI) m/z: 589 [⁸¹Br, (M+H)⁺], 587 [⁷⁹Br, (M+H)⁺].

Step 2: Compound 39-2

The compound obtained in step 1 (0.726 g, 1.24 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.344 g, 63%).

MS (APCI, ESI) m/z: 443 [⁸¹Br, (M+H)⁺], 441 [⁷⁹Br, (M+H)⁺].

Step 3: Compound 39-3

The compound obtained in step 2 (0.344 g, 0.779 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.248 g, 72%).

MS (APCI, ESI) m/z: 445 [⁸¹Br, (M+H)⁺], 443 [⁷⁹Br, (M+H)⁺].

Step 4: Compound 39-4

The compound obtained in step 3 (0.248 g, 0.559 mmol) was reacted in thesame manner as in step 9 of Example 3 to afford the desired compound(0.267 g, 90%).

MS (APCI, ESI) m/z: 529 [⁸¹Br, (M+H)⁺], 527 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 39-5

The compound obtained in step 4 (0.100 g, 0.190 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.201 g, 85%).

MS (APCI, ESI) m/z: 1255 (M+H)⁺.

Step 6: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyll-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-5-oxo-2-phenyl-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxo)methyl]phenyl}-L-alaninamide

The compound obtained in step 5 (0.201 g, 0.160 mmol) was reacted in thesame manner as in steps 11, 12, and 13 of Example 3 to afford thedesired compound (0.080 g, 36%).

MS (APCI, ESI) m/z: 1372 (M+H)⁺.

Example 40: Drug-Linker 38

Step 1: Compound 40-1

The compound obtained in step 4 of Example 4 (1.48 g, 2.24 mmol) and4-(dimethylamino)phenylboronic acid (0.741 g, 4.49 mmol) were reacted inthe same manner as in step 6 of Example 3 to afford the desired compound(0.520 g, 37%).

MS (APCI, ESI) m/z: 632 [⁸¹Br, (M+H)⁺], 630 [⁷⁹Br, (M+H)⁺].

Step 2: Compound 40-2

The compound obtained in step 1 (0.520 g, 0.825 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.167 g, 42%).

Step 3: Compound 40-3

The compound obtained in step 2 (0.167 g, 0.345 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.0650 g, 39%).

MS (APCI, ESI) m/z: 488 [⁸¹Br, (M+H)⁺], 486 [⁷⁹Br, (M+H)⁺].

Step 4: Compound 40-4

The compound obtained in step 3 (0.0650 g, 0.134 mmol) was reacted inthe same manner as in step 9 of Example 3 to afford the desired compound(0.0690 g, 90%).

MS (APCI, ESI) m/z: 572 [⁸¹Br, (M+H)⁺], 570 [⁷⁹Br, (M+H)⁺].

Step 5: Compound 40-5

The compound obtained in step 4 (0.0690 g, 0.121 mmol) was reacted inthe same manner as in step 10 of Example 3 to afford the desiredcompound (0.0660 g, 42%).

MS (APCI, ESI) m/z: 1297 (M+H)⁺.

Step 6: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-8′-[3-({(11aS)-2-[4-(dimethylamino)phenyl]-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-11′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 5 (0.0660 g, 0.0509 mmol) was reacted inthe same manner as in steps 11, 12, and 13 of Example 3 to afford thedesired compound (0.0350 g, 49%).

MS (APCI, ESI) m/z: 1417 (M+H)⁺.

Example 41: Drug-Linker 39

Step 1: N,N-Dimethylformamide adduct of5-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}valeric acid

In N,N-dimethylformamide (10 mL), 5-aminovaleric acid (0.436 g, 3.72mmol) was dissolved, to which 1-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxy}pyrrolidin-2,5-dione (1.36 g, 3.38 mmol) andtriethylamine (0.937 mL, 6.76 mmol) were added at room temperature, andthe resultant was stirred at room temperature for 1.5 hours. To thereaction solution, 1 N hydrochloric acid was added, which was extractedwith chloroform, and the organic layer obtained was washed with waterand brine and then dried over magnesium sulfate. After distillationunder reduced pressure, the resulting residue was purified by silica gelcolumn chromatography [organic layer for distribution withchloroform-chloroform:methanol:water=7:3:1 (v/v/v)] to afford thedesired compound (0.730 g, 45%) as a solid.

¹H-NMR (CDCl₃) δ: 8.06 (1H, s), 7.71 (1H, m), 7.54-7.52 (1H, m),7.46-7.32 (6H, m), 6.04 (1H, m), 5.18 (1H, m), 3.72 (1H, m), 3.17-3.10(2H, m), 3.00 (3H, s), 2.92 (3H, s), 2.84-2.80 (1H, m), 2.45-2.34 (3H,m), 2.28-2.24 (1H, m), 2.03-1.99 (1H, m), 1.66-1.58 (2H, m), 1.47-1.40(2H, m).

MS (APCI, ESI) m/z: 405 (M+H)⁺

Step 2: N-(5-{[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}pentanoyl)-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a‘-dihydro-1’H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 1 (0.0176 g, 0.0368 mmol) was reacted inthe same manner as in step 13 of Example 3 to afford the desiredcompound (0.00880 g, 18%).

MS (APCI, ESI) m/z: 1339 (M+H)⁺

Example 42: Drug-Linker 40

Step 1: Compound 42-1

The compound obtained in step 1 of Example 3 (5.00 g, 9.66 mmol) wasreacted in the same manner as in step 3 of Example 3 to afford thedesired compound (3.95 g, 100%). MS (APCI, ESI) m/z: 409 (M+H)⁺

Step 2: Compound 42-2

The compound obtained in step 1 (3.95 g, 9.67 mmol) was reacted in thesame manner as in step 2 of Example 24 to afford the desired compound(4.78 g, 87%).

MS (APCI, ESI) m/z: 565 (M+H)⁺

Step 3: Compound 42-3

The compound obtained in step 2 (4.78 g, 8.43 mmol) was reacted in thesame manner as in step 4 of Example 3 to afford the desired compound(2.36 g, 50%).

MS (APCI, ESI) m/z: 563 (M+H)⁺

Step 4: Compound 42-4

The compound obtained in step 3 (1.53 g, 2.72 mmol) was reacted in thesame manner as in step 5 of Example 3 to afford the desired compound(1.27 g, 69%).

¹H-NMR (CDCl₃) δ: 7.31 (2H, s), 7.15 (1H, m), 5.52 (1H, m), 4.65 (1H,m), 4.57 (1H, m), 3.95-3.89 (1H, m), 3.87 (3H, s), 3.75-3.58 (2H, m),3.18-3.14 (1H, m), 1.33-1.25 (3H, m), 1.10 (18H, m), 1.00-0.96 (2H, m),0.03 (9H, s).

Step 5: Compound 42-5

The compound obtained in step 4 (0.255 g, 0.367 mmol) and4-(aminomethyl)phenylboronic acid (0.344 g, 1.84 mmol) were reacted inthe same manner as in step 6 of Example 3 to afford the desired compound(0.148 g, 62%).

MS (APCI, ESI) m/z: 652 (M+H)⁺

Step 6: Compound 42-6

To a solution of the compound (0.142 g, 0.218 mmol) obtained in step 5and diprop-2-en-1-yl [(Z)-(methylsulfanyl)methylidene]biscarbamate(0.079 g, 0.079 mmol, WO 9920628) in dimethylformamide (2.2 mL),triethylamine (0.090 mL, 0.653 mmol) and mercury chloride (II) (0.083 g,0.305 mmol) were added at room temperature. After the reaction solutionwas stirred at room temperature for 30 minutes, the reaction solutionwas diluted by addition of ethyl acetate, and mercury salts were removedthrough filtration. The organic layer obtained was washed with 0.1 Nphosphate buffer and brine. The organic layer was dried over sodiumsulfate, and then distillated under reduced pressure. The resultingresidue was purified by silica gel column chromatography [hexane:ethylacetate=100:0 (v/v) to 60:40 (v/v)] to afford the desired compound(0.157 g, 83%).

MS (APCI, ESI) m/z: 863 (M+H)⁺

Step 7: Compound 42-7

To a solution of the compound obtained in step 6 (0.153 g, 0.177 mmol)in THF (1.8 mL), a 1 mol/L tetrahydrofuran solution oftetrabutylammonium fluoride (0.266 mL, 0.266 mmol) was added at roomtemperature. After stirring at room temperature for 50 minutes, 0.1 Nphosphate buffer (pH 7.0) was added to the reaction solution, which wasextracted with ethyl acetate, and the organic layer obtained was washedwith 0.1 N phosphate buffer (pH 7.0). The resultant was dried oversodium sulfate, and then distillated under reduced pressure. Theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)] to afford the desiredcompound (0.115 g, 92%).

MS (APCI, ESI) m/z: 706 (M+H)⁺

Step 8: Compound 42-8

To a solution of the compound obtained in step 7 (0.115 g, 0.163 mmol)in N,N-dimethylformamide (1.0 mL), 1,5-dibromopentane (0.088 mL, 0.652mmol) and cesium carbonate (0.032 g, 0.098 mmol) were added at roomtemperature. After stirring at room temperature for 2 hours, 0.1 Nphosphate buffer (pH 7.0) was added to the reaction solution, which wasextracted with ethyl acetate. The organic layer obtained was washed with0.1 N phosphate buffer (pH 7.0) and brine and dried over sodium sulfate,and then distillated under reduced pressure. The resulting residue waspurified by silica gel column chromatography [hexane:ethyl acetate=100:0(v/v) to 50:50 (v/v)] to afford the desired compound (0.111 g, 80%).

MS (APCI, ESI) m/z: 856 [⁸¹Br, (M+H)⁺], 854 [⁷⁹Br, (M+H)⁺]

Step 9: Compound 42-9

The compound obtained in step 8 (0.105 g, 0.123 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.036 g, 41%).

MS (APCI, ESI) m/z: 772 [⁸¹Br, (M+H)⁺], 770 [⁷⁹Br, (M+H)⁺]

Step 10: Compound 42-10

The compound obtained in step 9 (0.163 g, 0.169 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.132 g, 88%).

MS (APCI, ESI) m/z: 880 (M+H)⁺

Step 11: Compound 42-11

To a solution of the compound obtained in step 10 (0.13 g, 0.147 mmol),5-{[(prop-2-en-1-yloxy)carbonyl]amino}pentanoic acid (0.0386 g, 0.192mmol), and 1-hydroxybenzotriazole monohydrate (0.038 g, 0.251 mmol) indichloromethane (3 mL), triethylamine (0.035 mL, 0.251 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.048 g,0.251 mmol) were added at room temperature, and the resultant wasstirred at room temperature for 1 hour. To the reaction solution, 0.1 Nphosphate buffer (pH 7.0) was added, and the resultant was extractedwith ethyl acetate. The organic layer obtained was washed with 0.1 Nphosphate buffer (pH 7.0), and ethyl acetate was distilled off underreduced pressure. The resultant was azeotroped with toluene, and theresulting residue was purified by silica gel column chromatography[hexane:ethyl acetate=100:0 (v/v) to 50:50 (v/v)] to afford the desiredcompound (0.150 g, 95%).

MS (APCI, ESI) m/z: 1063 (M+H)⁺

Step 12: Compound 42-12

To a solution of the compound obtained in step 11 (0.150 g, 0.141 mmol)in N,N-dimethylformamide (3.5 mL), an aqueous solution of lithiumacetate (1.52 M, 0.088 mL) was added at room temperature. After stirringat room temperature for 1.5 hours, water was added to the reactionsolution, which was extracted with ethyl acetate. The organic layer waswashed with water, and then distillated under reduced pressure. Theresulting residue was purified by silica gel column chromatography[chloroform:methanol=100:0 (v/v) to 95:5 (v/v)] to afford the desiredcompound (0.135 g, 96%).

MS (APCI, ESI) m/z: 907 (M+H)⁺

Step 13: Compound 42-13

To a solution of the compound obtained in step 12 (0.048 g, 0.053 mmol)and the compound obtained in step 9 (0.034 g, 0.048 mmol) inN,N-dimethylformamide (0.5 mL), cesium carbonate (0.011 g, 0.034 mmol)was added at room temperature. Stirring was performed at roomtemperature for 1.5 hours, and then at 45° C. for 5 hours. To thereaction solution, 0.1 N phosphate buffer (pH 7.0) was added, and theresultant was extracted with ethyl acetate. The organic layer obtainedwas washed with 0.1 N phosphate buffer (pH 7.0) and dried over sodiumsulfate, and then distillated under reduced pressure. The resultingresidue was purified by silica gel column chromatography[chloroform:methanol=100:0 (v/v) to 97:3 (v/v)] to afford the desiredcompound (0.038 g, 52%).

MS (APCI, ESI) m/z: 1534 (M+H)⁺

Step 14: Compound 42-14

The compound obtained in step 13 (0.038 g, 0.247 mmol) was reacted inthe same manner as in step 11 of Example 3. After the reaction solutionwas subjected to liquid separation, the organic solvent was distilledoff under reduced pressure, and the resulting compound was directly usedfor the subsequent reaction.

MS (APCI, ESI) m/z: 1420 (M+H)⁺

Step 15: Compound 42-15

To a solution of the compound obtained in step 14 (0.034 g, 0.140 mmol)in dimethylformamide (2 mL), pyrrolidine (0.048 mL, 0.574 mmol) andtetrakis(triphenylphosphine)palladium (0) (0.0054 g, 0.0046 mmol) wereadded at room temperature, and the resultant was stirred at roomtemperature for 50 minutes. After the organic solvent was distilled offunder reduced pressure, the resulting residue was dissolved in dimethylsulfoxide and purified by reversed-phase column chromatography (column:Develosil Combi-RP-5 (Nomura Chemical Co., Ltd.), 20 mm×100 mm: a 0.1%formic acid aqueous solution:0.1% formic acid acetonitrilesolution=79.2:20.8 to 51.4:48.6, flow rate: 25 mL/min, temperature: 25°C.) to afford the desired compound (0.020 g, 72%).

MS (APCI, ESI) m/z: 1168 (M+H)⁺

Step 16: Compound 42-16

To a solution of the compound obtained in step 15 (0.002 g, 0.0017 mmol)in dimethylformamide (0.5 mL), triethylamine (0.007 mL, 0.051 mmol) andcommercially available 1-{[4-(11,12-didehydrobenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]oxopyrrolidin-2,5-dione (0.083 g, 0.0205 mmol)were added, and the resultant was stirred at room temperature for 10hours. The organic solvent was distilled off under reduced pressure, andthe resulting residue was dissolved in dimethyl sulfoxide and purifiedby reversed-phase column chromatography (column: Develosil Combi-RP-5(Nomura Chemical Co., Ltd.), 20 mm×100 mm: a 0.1% formic acid aqueoussolution:0.1% formic acid acetonitrile solution=62.5:37.5 to 34.7:65.3,flow rate: 25 mL/min, temperature: 25° C.) and silica gel columnchromatography [organic layer for distribution withchloroform-chloroform:methanol:water=7:3:1 (v/v/v)] to afford thedesired compound (0.033 g, 14%).

MS (APCI, ESI) m/z: 1455 (M+H)⁺

Example 43: Drug-Linker 41

Step 1: Compound 43-1

The compound obtained in step 4 of Example 4 (0.72 g, 1.09 mmol) and3-methoxyphenylboronic acid (0.332 g, 2.18 mmol) were reacted in thesame manner as in step 6 of Example 3 to afford the desired compound(0.526 g, 78%).

MS (APCI, ESI) m/z: 619 [⁸¹Br, (M+H)⁺], 617 [⁷⁹Br, (M+H)⁺]

Step 2: Compound 43-2

The compound obtained in step 1 (0.526 g, 0.851 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.195 g, 49%).

MS (APCI, ESI) m/z: 473 [⁸¹Br, (M+H)⁺], 471 [⁷⁹Br, (M+H)⁺]

Step 3: Compound 43-3

The compound obtained in step 2 (0.195 g, 0.414 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.195 g, quantitative).

MS (APCI, ESI) m/z: 475 [⁸¹Br, (M+H)⁺], 473 [⁷⁹Br, (M+H)⁺]

Step 4: Compound 43-4

The compound obtained in step 3 (0.413 mmol) was reacted in the samemanner as in step 9 of Example 3 to afford the desired compound (0.106g, 46%).

MS (APCI, ESI) m/z: 559 [⁸¹Br, (M+H)⁺], 557 [⁷⁹Br, (M+H)⁺]

Step 5: Compound 43-5

The compound obtained in step 4 (0.069 g, 0.124 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.128 g, 80%).

MS (APCI, ESI) m/z: 1284 (M+H)⁺

Step 6: Compound 43-6

The compound obtained in step 5 (0.128 g, 0.099 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.105 g, 90%).

MS (APCI, ESI) m/z: 1170 (M+H)⁺

Step 7: Compound 43-7

The compound obtained in step 6 (0.105 g, 0.089 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.072 g, 80%).

MS (APCI, ESI) m/z: 1002 (M+H)⁺

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-11′-hydroxy-7′-methoxy-8′-(3-{[(11aS)-7-methoxy-2-(3-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.072 g, 0.072 mmol) was reacted in thesame manner as in step 13 of Example 3 to afford the desired compound(0.053 g, 52%).

MS (APCI, ESI) m/z: 1403 (M+H)⁺

Example 44: Drug-Linker 42

Step 1: Compound 44-1

The compound obtained in step 4 of Example 4 (0.68 g, 1.03 mmol) and3,4-dimethoxyphenylboronic acid (0.375 g, 2.06 mmol) were reacted in thesame manner as in step 6 of Example 3 to afford the desired compound(0.506 g, 75%).

MS (APCI, ESI) m/z: 649 [⁸¹Br, (M+H)⁺], 647 [⁷⁹Br, (M+H)⁺]

Step 2: Compound 44-2

The compound obtained in step 1 (0.506 g, 0.781 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.199 g, 50%).

MS (APCI, ESI) m/z: 503 [⁸¹Br, (M+H)⁺], 501 [⁷⁹Br, (M+H)⁺]

Step 3: Compound 44-3

The compound obtained in step 2 (0.169 g, 0.337 mmol) was reacted in thesame manner as in step 8 of Example 3 to afford the desired compound(0.231 g, quantitative).

MS (APCI, ESI) m/z: 505 [⁸¹Br, (M+H)⁺], 503 [⁷⁹Br, (M+H)⁺]

Step 4: Compound 44-4

The compound obtained in step 3 (0.337 mmol) was reacted in the samemanner as in step 9 of Example 3 to afford the desired compound (0.170g, 86%).

MS (APCI, ESI) m/z: 589 [⁸¹Br, (M+H)⁺], 587 [⁷⁹Br, (M+H)⁺]

Step 5: Compound 44-5

The compound obtained in step 4 (0.076 g, 0.136 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.116 g, 71%).

MS (APCI, ESI) m/z: 1314 (M+H)⁺

Step 6: Compound 44-6

The compound obtained in step 5 (0.116 g, 0.088 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.108 g, quantitative).

MS (APCI, ESI) m/z: 1200 (M+H)⁺

Step 7: Compound 44-7

The compound obtained in step 6 (0.090 mol) was reacted in the samemanner as in step 12 of Example 3 to afford the desired compound (0.066g, 71%).

MS (APCI, ESI) m/z: 1032 (M+H)⁺

Step 8: N-[4-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11a′S)-8′-(3-{[(11aS)-2-(3,4-dimethoxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-11′-hydroxy-7′-methoxy-5′-oxo-11,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide

The compound obtained in step 7 (0.066 g, 0.064 mmol) was reacted in thesame manner as in step 13 of Example 3 to afford the desired compound(0.053 g, 58%).

MS (APCI, ESI) m/z: 1434 (M+H)⁺

Synthesis of Drug D Example 45: Drug 1

Step 1: Prop-2-en-1-yl(2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamate

The compound obtained in step 5 of Example 1 (4.59 g, 8.15 mmol) wasreacted in the same manner as in step 9 of Example 3 to afford thedesired compound (4.86 g, 92%).

¹H-NMR (CDCl₃) δ: 8.97 (1H, s), 7.77 (1H, s), 6.77 (1H, s), 5.97-5.94(1H, m), 5.39-5.21 (2H, m), 4.67-4.59 (3H, m), 4.00-3.98 (1H, m),3.74-3.66 (5H, m), 3.05-3.03 (1H, m), 2.30-2.28 (1H, m), 1.72-1.70 (1H,m), 1.30-1.27 (3H, m), 1.11-1.05 (18H, m), 0.99-0.91 (9H, m), 0.61-0.53(4H, m), 0.10-0.06 (6H, m).

MS (APCI, ESI) m/z: 647 (M+H)⁺

Step 2: Prop-2-en-1-yl(2-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxy-5-{[tri(propan-2-yl)silyl]oxy}phenyl)carbamate

The compound obtained in step 1 (4.86 g, 7.51 mmol) was reacted in thesame manner as in step 7 of Example 1 to afford the desired compound(3.42 g, 86%).

¹H-NMR (CDCl₃) δ: 8.52 (1H, s), 7.71 (1H, s), 6.77 (1H, s), 6.00-5.94(1H, m), 5.35-5.27 (2H, m), 4.65-4.64 (3H, m), 4.33-4.31 (1H, m),3.82-3.77 (5H, m), 3.68-3.66 (1H, m), 3.15-3.13 (1H, m), 1.89-1.86 (2H,m), 1.30-1.26 (3H, m), 1.14-1.10 (18H, m), 0.66-0.51 (4H, m).

MS (APCI, ESI) m/z: 533 (M+H)⁺

Step 3: Prop-2-en-1-yl(11a′S)-11′-hydroxy-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 2 (6.68 g, 12.5 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(6.44 g, 97%).

¹H-NMR (CDCl₃) δ: 7.20 (1H, s), 6.69 (1H, s), 5.89-5.78 (2H, m),5.18-5.15 (2H, m), 4.62-4.60 (1H, m), 4.49-4.47 (1H, m), 3.85 (3H, s),3.74-3.71 (1H, m), 3.59-3.57 (1H, m), 3.33-3.30 (2H, m), 2.43-2.40 (1H,m), 1.76-1.73 (1H, m), 1.28-1.20 (3H, m), 1.09-1.07 (18H, m), 0.74-0.65(4H, m).

MS (APCI, ESI) m/z: 531 (M+H)⁺

Step 4: Prop-2-en-1-yl(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-8′-{[tri(propan-2-yl)silyl]oxy}-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 3 (3.24 g, 6.10 mmol) was reacted in thesame manner as in step 9 of Example 1 to afford the desired compound(3.86 g, 98%).

¹H-NMR (CDCl₃) δ: 7.20 (1H, s), 6.67 (1H, s), 6.01-5.98 (1H, m),5.79-5.73 (1H, m), 5.14-5.10 (2H, m), 4.64-4.61 (1H, m), 4.37-4.34 (1H,m), 3.86 (3H, s), 3.72-3.69 (1H, m), 3.52-3.50 (1H, m), 3.29-3.26 (1H,m), 2.38-2.34 (1H, m), 1.55-1.51 (1H, m), 1.28-1.24 (3H, m), 1.15-1.07(18H, m), 0.81-0.66 (13H, m), 0.21 (3H, s), 0.18 (3H, s).

MS (APCI, ESI) m/z: 645 (M+H)⁺

Step 5: Prop-2-en-1-yl(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-8′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 4 (4.49 g, 6.96 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(3.24 g, 95%).

¹H-NMR (CDCl₃) δ: 7.25 (1H, s), 6.73 (1H, s), 6.02-6.00 (1H, m), 5.91(1H, s), 5.77-5.75 (1H, m), 5.11-5.09 (2H, m), 4.64-4.62 (1H, m),4.41-4.40 (1H, m), 3.95 (3H, s), 3.72-3.70 (1H, m), 3.54-3.53 (1H, m),3.29-3.26 (1H, m), 2.36-2.34 (1H, m), 1.56-1.54 (1H, m), 0.79-0.67 (13H,m), 0.21 (3H, s), 0.20 (3H, s).

MS (APCI, ESI) m/z: 489 (M+H)⁺

Step 6: Prop-2-en-1-yl(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 5 (0.080 g, 0.164 mmol) was reacted in thesame manner as in step 10 of Example 3 to afford the desired compound(0.160 g, 98%).

¹H-NMR (DMSO-D₆) δ: 7.44-7.42 (3H, m), 7.12-7.10 (2H, m), 7.05-7.03 (1H,m), 6.92-6.90 (2H, m), 6.61-6.59 (1H, m), 5.87-5.81 (3H, m), 5.10-5.07(4H, m), 4.66-4.55 (3H, m), 4.43-4.39 (2H, m), 4.21-3.94 (5H, m), 3.83(3H, s), 3.81 (3H, s), 3.76 (3H, s), 3.65-3.62 (1H, m), 3.56-3.54 (1H,m), 3.42-3.39 (1H, m), 3.22-3.14 (2H, m), 2.77-2.73 (1H, m), 2.42-2.33(1H, m), 1.81-1.79 (4H, m), 1.55-1.44 (3H, m), 0.82 (9H, s), 0.72-0.53(4H, m), 0.19 (3H, s), 0.17 (3H, s).

MS (APCI, ESI) m/z: 993 (M+H)⁺

Step 7: Prop-2-en-1-yl(11a′S)-11′-hydroxy-7′-methoxy-8′-{[5-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)pentyl]oxy}-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 6 (160 mg, 0.161 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(141 mg, quantitative).

¹H-NMR (DMSO-D₆) δ: 7.44-7.42 (3H, m), 7.08-7.06 (3H, m), 6.92-6.90 (2H,m), 6.82-6.79 (1H, m), 6.56-6.54 (1H, m), 5.77-5.74 (3H, m), 5.09 (4H,s), 4.58-4.55 (3H, m), 4.43-4.41 (2H, m), 4.16-4.01 (5H, m), 3.81-3.81(6H, m), 3.76 (3H, s), 3.64 (1H, s), 3.56-3.53 (1H, m), 3.42-3.38 (1H,m), 3.25-3.13 (2H, m), 2.74-2.70 (1H, m), 2.37-2.34 (1H, m), 1.82-1.79(4H, m), 1.59-1.56 (3H, m), 0.66-0.62 (4H, m).

MS (APCI, ESI) m/z: 879 (M+H)⁺

Step 8:(11a′S)-7′-Methoxy-8′-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 7 (141 mg, 0.161 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(109.8 mg, 99%).

¹H-NMR (DMSO-D₆) δ: 7.92-7.91 (1H, m), 7.45 (1H, s), 7.39-7.37 (2H, m),7.33 (1H, s), 7.29 (1H, s), 6.92-6.89 (2H, m), 6.85 (1H, s), 6.56-6.54(1H, m), 6.31 (1H, s), 4.19-4.12 (2H, m), 4.05-3.99 (1H, m), 3.95-3.93(2H, m), 3.82-3.79 (4H, m), 3.76 (3H, s), 3.66 (3H, s), 3.52-3.46 (3H,m), 3.30-3.21 (2H, m), 2.78-2.74 (1H, m), 2.45-2.42 (1H, m), 2.06-2.05(1H, m), 1.89-1.82 (4H, m), 1.60-1.58 (2H, m), 0.80-0.63 (4H, m).

MS (APCI, ESI) m/z: 693 (M+H)⁺

Example 46: Drug 2

Step 1: Compound 46-1

The compound obtained in step 10 of Example 26 (0.246 g, 0.514 mmol) wasused and reacted in the same manner as in step 1 of Example 4 to affordthe desired compound (0.122 g, 40%).

MS (APCI, ESI) m/z: 601 [⁸¹Br, (M+H)⁺], 599 [⁷⁹Br, (M+H)⁺].

Step 2: Compound 46-2

The compound obtained in step 1 (0.122 g, 0.204 mmol) was reacted in thesame manner as in step 6 of Example 45 to afford the desired compound(0.176 g, 86%).

Step 3: Compound 46-3

The compound obtained in step 2 (0.0870 g, 0.0864 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.0660 g, 86%). MS (APCI, ESI) m/z: 892 (M+H)⁺

Step 4:4-[(11aS)-7-Methoxy-8-(3-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}propoxy)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl]benzylacetate

The compound obtained in step 3 (0.0660 g, 0.0793 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0444 g, 85%).

¹H-NMR (CDCl₃) δ: 7.80-7.79 (1H, m), 7.66-7.65 (1H, m), 7.52-7.50 (2H,m), 7.39-7.33 (5H, m), 6.86-6.85 (1H, m), 6.14-6.13 (1H, m), 5.09 (2H,s), 4.32-4.2 (6H, m), 3.95-3.95 (3H, m), 3.85-3.82 (4H, m), 3.71-3.68(2H, m), 3.55-3.49 (3H, m), 3.42-3.33 (1H, m), 2.79-2.72 (1H, m),2.54-2.50 (1H, m), 2.40-2.36 (2H, m), 2.02-1.98 (1H, m), 1.26-1.24 (1H,m), 0.76-0.72 (4H, m).

MS (APCI, ESI) m/z: 706 (M+H)⁺

Example 47: Drug 3

Step 1:[4-(Benzyl)-5-methoxy-2-nitrophenyl][(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]methanone

To a solution of the compound obtained in step 1 of Example 37 (6.49 g,14.7 mmol) in tetrahydrofuran (147 mL), lithium borohydride (0.642 g,29.5 mmol) was added at 0° C., and the resultant was stirred at roomtemperature for 2 hours. To the reaction solution, 1 N hydrochloric acidwas added, which was extracted with ethyl acetate. The organic layerobtained was washed with brine and dried over magnesium sulfate, andthen distillated under reduced pressure. The resulting residue (6.94 g,quantitative) was used for the subsequent step without purification.

MS (APCI, ESI) m/z: 413 (M+H)⁺

Step 2:(6S)-5-[4-(Benzyloxy)-5-methoxy-2-nitrobenzoyl]-5-azaspiro[2.4]heptane-6-carbaldehyde

The compound obtained in step 1 (4.50 g, 11.0 mmol) was reacted in thesame manner as in step 8 of Example 1 to afford the desired compound(1.94 g, 43%).

MS (APCI, ESI) m/z: 411 (M+H)⁺

Step 3:(11a′S)-8′-Hydroxy-7′-methoxy-1′,10′,11′,11a′-tetrahydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

To a mixed solution of the compound obtained in step 2 (1.94 g, 4.73mmol) in tetrahydrofuran (25 mL), ethyl acetate (25 mL), and methanol(25 mL), 5% palladium carbon (moisture content: 54%, 1.0 g) was addedunder the nitrogen atmosphere, and the reaction solution was thenstirred under the hydrogen atmosphere at room temperature for 22 hours.After the reaction solution was filtered through a Celite, the filtratewas distillated under reduced pressure. The resulting residue waspurified by silica gel column chromatography [hexane:ethyl acetate=80:20(v/v) to 0:100 (v/v)] to afford the desired compound (1.20 g, 93%).

¹H-NMR (CDCl₃) δ: 7.55 (1H, s), 6.16 (1H, s), 5.86 (1H, s), 4.08-4.02(2H, m), 3.86 (3H, s), 3.72-3.69 (1H, m), 3.57-3.37 (3H, m), 2.04-2.01(1H, m), 1.78-1.75 (1H, m), 0.79-0.53 (4H, m).

MS (APCI, ESI) m/z: 275 (M+H)⁺

Step 4: Prop-2-en-1-yl(11a′S)-8′-[(5-bromopentyl)oxy]-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 3 of Example 45 (0.300 g, 0.614 mmol) wasreacted in the same manner as in step 2 of Example 3 to afford thedesired compound (0.388 g, 99%).

¹H-NMR (CDCl₃) δ: 7.24 (1H, s), 6.60 (1H, s), 6.02-5.98 (1H, m),5.80-5.75 (1H, m), 5.11-5.06 (2H, m), 4.68-4.64 (1H, m), 4.40-4.38 (1H,m), 4.02-3.98 (2H, m), 3.92 (3H, s), 3.72-3.69 (1H, m), 3.54-3.52 (1H,m), 3.46-3.41 (2H, m), 3.29-3.26 (1H, m), 2.38-2.34 (1H, m), 1.94-1.87(4H, m), 1.65-1.62 (2H, m), 1.55-1.55 (1H, m), 0.86 (9H, s), 0.75-0.67(4H, m), 0.24-0.22 (6H, m).

MS (APCI, ESI) m/z: 639 [⁸¹Br, (M+H)⁺], 637 [⁷⁹Br, (M+H)⁺].

Step 5: Prop-2-en-1-yl(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 4 (0.203 g, 0.318 mmol) was reacted withthe compound obtained in step 3 (0.131 g, 0.478 mmol) in the same manneras in step 10 of Example 3 to afford the desired compound (0.0880 g,33%).

MS (APCI, ESI) m/z: 831 (M+H)⁺

Step 6: Prop-2-en-1-yl(11a′S)-11′-hydroxy-7′-methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,10′,11′,11a′-tetrahydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 5 (0.0880 g, 0.106 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.0500 g, 66%).

MS (APCI, ESI) m/z: 717 (M+H)⁺

Step 7:(11a′S)-7′-Methoxy-8′-[(5-{[(11a′S)-7′-methoxy-5′-oxo-5′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-8′-yl]oxy}pentyl)oxy]-1′,10′,11′,11a′-tetrahydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 6 (0.0500 g, 0.0698 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0330 g, 77%).

¹H-NMR (CDCl₃) δ: 7.80 (1H, m), 7.58 (1H, s), 7.52 (1H, s), 6.81 (1H,s), 6.05 (1H, s), 4.17-3.97 (5H, m), 3.94 (3H, s), 3.87 (1H, m), 3.84(3H, s), 3.72-3.68 (3H, m), 3.51-3.45 (5H, m), 2.54-2.51 (1H, m),2.03-1.90 (6H, m), 1.75-1.68 (2H, m), 0.66 (8H, m).

MS (APCI, ESI) m/z: 615 (M+H)⁺

Example 48: Drug 4

Step 1: Compound 48-1

The compound obtained in step 4 of Example 39 (0.165 g, 0.313 mmol) wasreacted in the same manner as in step 6 of Example 45 to afford thedesired compound (0.270 g, 92%).

MS (APCI, ESI) m/z: 935 (M+H)⁺.

Step 2: Compound 48-2

The compound obtained in step 1 (0.270 g, 0.289 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.208 g, 88%).

MS (APCI, ESI) m/z: 821 (M+H)⁺.

Step 3:(11a′S)-7′-Methoxy-8′-(3-{[(11aS)-7-methoxy-5-oxo-2-phenyl-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-11′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 2 (0.208 g, 0.253 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.130 g, 80%).

MS (APCI, ESI) m/z: 635 (M+H)⁺.

Example 49: Drug 5

Step 1: Compound 49-1

Compound 4-1 (8.52 g, 13.2 mmol) and compound 37-5 (6.09 g, 14.5 mmol),obtained in step 4 of Example 37, were used and reacted in the samemanner as in step 10 of Example 3 to afford the desired compound (10.7g, 83%).

¹H-NMR (CDCl₃) δ: 7.36 (1H, s), 7.34 (1H, s), 7.26 (1H, s), 7.23 (1H,s), 5.53-5.46 (2H, m), 4.72-4.69 (2H, m), 4.59-4.55 (1H, m), 4.28-4.20(6H, m), 3.90-3.89 (6H, m), 3.80-3.63 (6H, m), 3.57-3.54 (1H, m),3.42-3.39 (1H, m), 2.87-2.82 (1H, m), 2.47-2.40 (3H, m), 2.24-2.20 (1H,m), 2.04-1.99 (1H, m), 0.99-0.95 (4H, m), 0.87 (9H, s), 0.70-0.63 (4H,m), 0.09 (6H, s), 0.02-0.00 (18H, m).

Step 2: Compound 49-2

The compound obtained in step 1 (10.7 g, 10.9 mmol) was reacted in thesame manner as in step 3 of Example 3 to afford the desired compound(9.45 g, 100%).

¹H-NMR (CDCl₃) δ: 7.36-7.34 (2H, m), 7.26 (1H, s), 7.23 (1H, s),5.53-5.47 (2H, m), 4.72-4.64 (3H, m), 4.30-4.20 (6H, m), 3.89 (6H, s),3.85-3.62 (7H, m), 3.42-3.39 (1H, m), 2.99-2.93 (1H, m), 2.47-2.39 (3H,m), 2.25-2.07 (3H, m), 0.99-0.95 (4H, m), 0.89-0.86 (1H, m), 0.70-0.64(3H, m), 0.02-0.00 (18H, m).

Step 3: Compound 49-3

The compound obtained in step 2 (9.45 g, 10.9 mmol) was reacted in thesame manner as in step 4 of Example 3 to afford the desired compound(9.14 g, 97%).

¹H-NMR (CDCl₃) δ: 7.37 (1H, s), 7.33 (1H, s), 7.27-7.26 (2H, m),5.54-5.51 (2H, m), 4.77-4.70 (2H, m), 4.64-4.61 (1H, m), 4.31-4.20 (6H,m), 3.91 (3H, s), 3.90 (3H, s), 3.80-3.65 (6H, m), 3.60-3.55 (1H, m),3.42-3.39 (1H, m), 2.83-2.75 (1H, m), 2.47-2.41 (3H, m), 2.25-2.20 (1H,m), 1.00-0.95 (4H, m), 0.92-0.85 (1H, m), 0.71-0.63 (3H, m), 0.02-0.01(18H, m).

Step 4: Compound 49-4

The compound obtained in step 3 (4.27 g, 4.94 mmol) was reacted in thesame manner as in step 5 of Example 3 to afford the desired compound(3.16 g, 64%).

¹H-NMR (CDCl₃) δ: 7.37 (1H, s), 7.32 (1H, s), 7.27-7.25 (2H, m),7.15-7.14 (1H, m), 5.53-5.50 (2H, m), 4.77-4.70 (2H, m), 4.64-4.60 (1H,m), 4.30-4.20 (6H, m), 3.90 (6H, s), 3.81-3.66 (4H, m), 3.42-3.39 (1H,m), 3.18-3.13 (1H, m), 2.47-2.41 (3H, m), 2.25-2.20 (1H, m), 0.99-0.94(4H, m), 0.89-0.86 (1H, m), 0.69-0.64 (3H, m), 0.02-0.00 (18H, m).

Step 5: Compound 49-5

The compound obtained in step 4 (1.00 g, 1.00 mmol) was reacted in thesame manner as in step 6 of Example 3 to afford the desired compound(0.900 g, 94%).

¹H-NMR (CDCl₃) δ: 7.69-7.65 (1H, m), 7.57-7.53 (1H, m), 7.49-7.45 (1H,m), 7.39-7.36 (4H, m), 7.33 (1H, s), 7.27 (1H, s), 6.90-6.88 (2H, m),5.53-5.50 (2H, m), 4.77-4.71 (2H, m), 4.62-4.59 (1H, m), 4.30-4.20 (5H,m), 3.91 (3H, s), 3.91 (3H, s), 3.82 (3H, s), 3.80-3.65 (4H, m),3.42-3.39 (1H, m), 3.16-3.10 (1H, m), 2.47-2.41 (3H, m), 2.25-2.20 (1H,m), 1.00-0.96 (4H, m), 0.89-0.85 (1H, m), 0.70-0.63 (3H, m), 0.02-0.01(18H, m).

Step 6:(11a′S)-7′-Methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

To a solution of the compound obtained in step 5 (0.900 g, 0.942 mmol)in THF (30 mL) and ethanol (3 mL), lithium borohydride (0.456 g, 18.8mmol) was slowly added at 0° C. After the reaction mixture was stirredat room temperature for 1.5 hours, water was added to the reactionmixture, which was vigorously stirred. The reaction mixture wasextracted with chloroform. The organic layer was distillated underreduced pressure, and the resulting residue was dissolved indichloromethane (10 mL), ethanol (20 mL), and water (5 mL). Silica gel(15.0 g) was added to the reaction mixture, which was stirred under thenitrogen atmosphere for 3 days. The reaction mixture was filtered, andthe organic layer was washed with brine and dried over anhydrous sodiumsulfate. After filtration followed by distillation under reducedpressure, the resulting residue was purified by silica gel columnchromatography [chloroform:methanol=100:0 (v/v) to 92:8 (v/v)] to affordthe desired compound (0.308 g, 49%).

MS (APCI, ESI) m/z: 663 (M+H)⁺

Example 50: Drug 6

Step 1: Di-2-propen-1-yl {1,5-pentanediylbis[oxy(6-{[(6S)-6-(hydroxymethyl)-5-azaspiro[2.4]hept-5-yl]carbonyl}-4-methoxybenzen-3,1-diyl)]}biscarbamate

Bisallyloxycarbonyl form 15-6 (0.460 g, 0.508 mmol), obtained in step 5of Example 15, was dissolved in methanol (10 mL), and potassiumcarbonate (351 mg, 2.54 mmol) was then added thereto, and the resultantwas stirred at room temperature for 30 minutes. Thereto, 50 mL of asaturated aqueous ammonium chloride was added, and the resultant wasextracted with ethyl acetate. The organic layer was dried over anhydroussodium sulfate. The resultant was distillated under reduced pressure toafford the desired compound (0.421 g, quantitative).

¹H-NMR (DMSO-D₆) δ: 9.19 (2H, s), 7.22 (2H, s), 6.89 (2H, s), 5.97-5.92(2H, m), 5.33 (2H, m), 5.22 (2H, m), 4.81 (2H, m), 4.55 (4H, m), 4.26(2H, s), 3.96 (4H, m), 3.74 (6H, s), 3.62 (2H, m), 3.56 (2H, s), 3.37(2H, m), 3.11 (2H, m), 1.88-1.78 (8H, m), 1.56-1.54 (2H, m), 0.54-0.43(8H, m).

MS (APCI, ESI) m/z: 821 (M+H)⁺.

Step 2: Diprop-2-en-1-yl(11a′S,11a″″S)-8′,8″-[pentan-1,5-diylbis(oxy)]bis(11′-hydroxy-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate)

The compound obtained in step 1 (0.421 g, 0.513 mmol) was reacted in thesame manner as in step 8 of Example 15 to afford the desired compound(0.326 g, 78%).

¹H-NMR (DMSO-D₆) δ: 7.07 (2H, s), 6.80 (2H, s), 6.55 (2H, m), 5.84-5.81(2H, m), 5.75 (2H, m), 5.09-5.05 (4H, m), 4.62 (2H, mz), 4.40 (2H, m),3.98 (4H, m), 3.81 (6H, s), 3.54 (2H, m), 3.43-3.37 (2H, m), 3.14 (2H,m), 2.35 (2H, m), 1.81-1.79 (4H, m), 1.59-1.56 (4H, m), 0.70-0.59 (8H,m).

MS (APCI, ESI) m/z: 817 (M+H)⁺.

Step 3:(11a′S,11a″″S)-8′,8″-[1,5-Pentanediylbis(oxy)]bis(7′-methoxy-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one)

The compound obtained in step 2 (0.326 g, 0.399 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.208 g, 85%).

¹H-NMR (DMSO-D₆) δ: 7.91 (2H, m), 7.32 (2H, s), 6.84 (2H, s), 4.11 (2H,m), 4.06 (2H, m), 3.82 (6H, s), 3.51-3.31 (6H, m), 2.43 (2H, m), 2.05(2H, m), 1.82-1.80 (4H, m), 1.60-1.58 (2H, m), 0.79-0.77 (2H, m),0.68-0.64 (6H, m).

MS (APCI, ESI) m/z: 613 (M+H)⁺.

Example 51: Drug 7

Step 1: Compound 51-1

Compound 46-2 (0.0870 g, 0.0863 mmol) was reacted in the same manner asin step 1 of Example 27 to afford the desired compound (0.0660 mg, 79%).

¹H-NMR (CDCl₃) δ: 7.50 (11H, s), 7.36-7.34 (4H, m), 7.27-7.24 (2H, m),6.80 (1H, s), 6.64 (1H, s), 6.03 (1H, m), 5.80-5.74 (2H, m), 5.10-5.06(3H, m), 4.70 (2H, m), 4.67-4.60 (2H, m), 4.41-4.37 (3H, m), 4.23-4.20(6H, m), 3.90 (3H, s), 3.89 (3H, s), 3.72 (1H, m), 3.65-3.62 (1H, m),3.51 (1H, m), 3.34-3.26 (2H, m), 2.75-2.71 (1H, m), 2.39-2.35 (3H, m),1.74 (1H, m), 1.56-1.52 (1H, m), 0.85 (9H, s), 0.80-0.62 (4H, m), 0.22(3H, s), 0.21 (3H, s).

MS (APCI, ESI) m/z: 965 (M+H)⁺.

Step 2: Compound 51-2

The compound obtained in step 1 (0.0660 g, 0.0683 mmol) was reacted inthe same manner as in step 11 of Example 3 to afford the desiredcompound (0.0590 g, quantitative).

MS (APCI, ESI) m/z: 851 (M+H)⁺.

Step 3:(11a′S)-8′-[3-({(11aS)-2-[4-(Hydroxymethyl)phenyl]-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-7′-methoxy-11′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 2 (0.0590 g, 0.0693 mmol) was reacted inthe same manner as in step 12 of Example 3 to afford the desiredcompound (0.0290 g, 63%).

¹H-NMR (CDCl₃) δ: 7.80-7.79 (1H, m), 7.64-7.64 (1H, m), 7.52-7.51 (2H,m), 7.36-7.33 (4H, m), 6.85 (1H, m), 6.14 (1H, m), 4.69 (2H, m),4.30-4.26 (6H, m), 3.95-3.95 (3H, m), 3.86-3.84 (3H, m), 3.67 (1H, m),3.56-3.54 (2H, m), 3.50-3.48 (2H, m), 3.39-3.30 (1H, m), 2.80-2.73 (1H,m), 2.52 (1H, m), 2.41-2.39 (2H, m), 2.00 (1H, m), 1.73-1.72 (1H, m),0.76-0.70 (4H, m).

MS (APCI, ESI) m/z: 665 (M+H)⁺.

Example 52: Drug 8

Step 1:(11aS)-7-Methoxy-2-(4-methoxyphenyl)-10-{[2-(trimethylsilyl)ethoxy]methyl}-8-{[tri(propan-2-yl)silyl]oxy}-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5,11(10H,11aH)-dione

The compound obtained in step 4 of Example 42 (0.519 g, 0.747 mmol) wasreacted in the same manner as in step 6 of Example 3 to afford thedesired compound (0.511 g, quantitative).

¹H-NMR (CDCl₃) δ: 7.41-7.31 (5H, m), 6.91-6.85 (2H, m), 5.52 (1H, m),4.64 (1H, m), 4.57 (1H, m), 3.97-3.90 (1H, m), 3.88 (3H, s), 3.83 (3H,s), 3.75-3.56 (2H, m), 3.19-3.09 (1H, m), 1.36-1.23 (3H, m), 1.11 (18H,m), 1.02-0.97 (2H, m), 0.03 (9H, s).

MS (APCI, ESI) m/z: 653[(M+H)⁺]

Step 2:(11aS)-7-Methoxy-2-(4-methoxyphenyl)-8-{[tri(propan-2-yl)silyl]oxy}-1,11a-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 1 (0.178 g, 0.272 mmol) was reacted in thesame manner as in step 7 of Example 3 to afford the desired compound(0.094 g, 68%).

¹H-NMR (CDCl₃) δ: 7.87 (1H, m), 7.51 (1H, s), 7.41-7.39 (1H, m),7.36-7.33 (2H, m), 6.93-6.89 (2H, m), 6.86 (1H, s), 4.44-4.38 (1H, m),3.90 (3H, s), 3.83 (3H, s), 3.61-3.53 (1H, m), 3.41-3.34 (1H, m),1.33-1.25 (3H, m), 1.11-1.06 (18H, m).

MS (APCI, ESI) m/z: 507 (M+H)⁺

Step 3:(11aS)-7-Methoxy-2-(4-methoxyphenyl)-8-{[tri(propan-2-yl)silyl]oxy}-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one

The compound obtained in step 2 (0.063 g, 0.124 mmol) was used andreacted in the same manner as in step 8 of Example 3 to afford thedesired compound (0.046 g, 72%).

¹H-NMR (CDCl₃) δ: 7.53-7.48 (2H, m), 7.33-7.29 (2H, m), 6.90-6.86 (2H,m), 6.13-6.11 (1H, m), 4.36-4.29 (1H, m), 4.11 (1H, s), 3.82 (3H, s),3.79 (3H, s), 3.59-3.50 (2H, m), 3.40-3.31 (1H, m), 2.78-2.68 (1H, m),1.31-1.20 (3H, m), 1.13-1.02 (18H, m).

MS (APCI, ESI) m/z: 509 (M+H)⁺

Step 4: Prop-2-en-1-yl(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-8-{[tri(propan-2-yl)silyl]oxy}-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 3 (0.046 g, 0.090 mmol) was used andreacted in the same manner as in step 9 of Example 3 to afford thedesired compound (0.03 g, 56%).

¹H-NMR (CDCl₃) δ: 7.39-7.36 (1H, m), 7.31-7.28 (2H, m), 7.22 (1H, s),6.90-6.86 (3H, m), 6.75-6.72 (1H, m), 5.82-5.69 (1H, m), 5.18-5.08 (2H,m), 4.59-4.52 (1H, m), 4.48-4.39 (1H, m), 4.39-4.29 (1H, m), 4.23-4.12(1H, m), 3.86 (3H, s), 3.82 (3H, s), 3.64-3.58 (1H, m), 3.32-3.25 (1H,m), 2.73-2.65 (1H, m), 1.30-1.20 (2H, m), 1.12-1.06 (18H, m).

MS (APCI, ESI) m/z: 593 (M+H)⁺

Step 5: Prop-2-en-1-yl(11aS)-8-hydroxy-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-10(5H)-carboxylate

The compound obtained in step 4 (0.030 g, 0.050 mmol) was reacted in thesame manner as in step 10 of Example 1 to afford the desired compound(0.015 g, 0.034 mmol).

¹H-NMR (CDCl₃) δ: 7.39-7.25 (4H, m), 6.92-6.78 (3H, m), 6.03-5.92 (1H,m), 5.86-5.68 (1H, m), 5.20-5.07 (2H, m), 4.66-4.57 (1H, m), 4.52-4.40(1H, m), 4.40-4.27 (1H, m), 4.27-4.16 (1H, m), 3.95 (3H, s), 3.82 (3H,s), 3.66-3.59 (1H, m), 3.32-3.21 (1H, m), 2.74-2.64 (1H, m).

MS (APCI, ESI) m/z: 437 (M+H)⁺

Step 6: Prop-2-en-1-yl(11a′S)-8′-(3-bromopropoxy)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 5 of Example 45 (0.131 g, 0.268 mmol) wasreacted in the same manner as in step 1 of Example 4 to afford thedesired compound (0.086 g, 52%).

¹H-NMR (CDCl₃) δ: 7.24 (1H, s), 6.65 (1H, s), 6.02 (1H, m), 5.87-5.71(1H, m), 5.15-5.04 (2H, m), 4.72-4.62 (1H, m), 4.44-4.32 (1H, m),4.23-4.07 (3H, m), 3.92 (3H, s), 3.77-3.47 (4H, m), 3.28 (1H, m), 2.37(3H, m), 1.57-1.52 (1H, m), 0.86 (9H, s), 0.82-0.57 (4H, m), 0.21 (6H,m).

MS (APCI, ESI) m/z: 611 [⁸¹Br, (M+H)⁺], 609 [⁷⁹Br, (M+H)⁺]

Step 7: Prop-2-en-1-yl(11a′S)-11′-{[tert-butyl(dimethyl)silyl]oxy}-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 5 (0.015 g, 0.034 mmol) and the compoundobtained in step 6 (0.030 g, 0.048 mmol) were reacted in the same manneras in step 10 of Example 3 to afford the desired compound (0.032 g,96%).

MS (APCI, ESI) m/z: 965 (M+H)⁺

Step 8: Prop-2-en-1-yl(11a′S)-11′-hydroxy-7′-methoxy-8′-[3-({(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-10-[(prop-2-en-1-yloxy)carbonyl]-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl}oxy)propoxy]-5′-oxo-11′,11a′-dihydro-1′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-10′(5′H)-carboxylate

The compound obtained in step 7 (0.031 g, 0.032 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.026 g, 95%).

MS (APCI, ESI) m/z: 851 (M+H)⁺

Step 9:(11a′S)-7′-Methoxy-8′-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 8 (0.026 g, 0.030 mmol) was reacted in thesame manner as in step 12 of Example 3 to afford the desired compound(0.018 g, 88%).

¹H-NMR (CDCl₃) δ: 7.80 (1H, m), 7.54-7.51 (3H, m), 7.33-7.29 (2H, m),6.91-6.85 (3H, m), 6.14 (1H, s), 4.35-4.17 (6H, m), 3.95 (3H, s), 3.85(3H, s), 3.82 (3H, s), 3.76-3.25 (5H, m), 2.79-2.69 (1H, m), 2.52 (1H,m), 2.45-2.35 (1H, m), 2.03-1.96 (1H, m), 1.28-1.23 (2H, m), 0.78-0.69(4H, m).

MS (APCI, ESI) m/z: 665 (M+H)⁺

Example 53: Drug 9

Step 1: Compound 53-1

The compound obtained in step 4 of Example 43 (0.027 g, 0.048 mmol) wasreacted in the same manner as in step 6 of Example 45 to afford thedesired compound (0.037 g, 79%).

MS (APCI, ESI) m/z: 965 (M+H)⁺

Step 2: Compound 53-2

The compound obtained in step 1 (0.037 g, 0.038 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(35 mg, quantitative).

MS (APCI, ESI) m/z: 851[(M+H)⁺]

Step 3:(11a′S)-7′-Methoxy-8′-(3-{[(11aS)-7-methoxy-2-(3-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 2 (0.038 mmol) was reacted in the samemanner as in steps 6 to 8 of Example 45 to afford the desired compound(25 mg, 99%).

¹H-NMR (CDCl₃) δ: 7.81-7.78 (1H, m), 7.65-7.62 (1H, m), 7.53-7.49 (2H,m), 7.24 (1H, m), 6.96 (1H, m), 6.91-6.88 (1H, m), 6.85 (1H, m), 6.78(1H, m), 6.14 (1H, m), 4.41-4.18 (5H, m), 3.97-3.92 (3H, m), 3.88-3.84(3H, m), 3.83 (3H, s), 3.76-3.25 (5H, m), 2.78-2.71 (1H, m), 2.52 (1H,m), 2.45-2.35 (2H, m), 2.03-1.96 (1H, m), 1.29-1.21 (2H, m), 0.78-0.69(4H, m).

MS (APCI, ESI) m/z: 664 (M+H)⁺

Example 54: Drug 10

Step 1: Compound 54-1

The compound obtained in step 4 of Example 44 (0.027 g, 0.046 mmol) wasreacted in the same manner as in step 6 of Example 45 to afford thedesired compound (0.037 g, 81%).

MS (APCI, ESI) m/z: 995 (M+H)⁺

Step 2: Compound 54-2

The compound obtained in step 1 (0.037 g, 0.037 mmol) was reacted in thesame manner as in step 11 of Example 3 to afford the desired compound(0.034 g, quantitative).

MS (APCI, ESI) m/z: 881 (M+H)⁺

Step 3:(11a′S)-8′-(3-{[(11aS)-2-(3,4-Dimethoxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-7′-methoxy-1′,11a′-dihydro-5′H-spiro[cyclopropane-1,2′-pyrrolo[2,1-c][1,4]benzodiazepine]-5′-one

The compound obtained in step 2 (0.037 mmol) was reacted in the samemanner as in step 12 of Example 3 to afford the desired compound (0.027g, quantitative).

¹H-NMR (CDCl₃) δ: 7.81-7.78 (1H, m), 7.55-7.49 (3H, m), 6.99-6.96 (1H,m), 6.87-6.82 (3H, m), 6.14 (1H, m), 4.41-3.28 (22H, m), 2.77-2.71 (1H,m), 2.57-2.48 (1H, m), 2.45-2.34 (2H, m), 2.04-1.96 (1H, m), 1.43-1.11(2H, m), 0.79-0.67 (4H, m).

MS (APCI, ESI) m/z: 695 (M+H)⁺

Synthesis of Glycan Donor Example 55: [N₃-PEG (3)]₂-SG (10)-Ox

(FIG. 51 , the schematic diagram in the right of the structural formularepresents the corresponding structure in the schematic diagram of anintermediate having a linker structure to which an azide group has beenintroduced as represented by the reaction formula of Example 58.)

Step 1: [N₃-PEG (3)]₂-SG (10)

Into a 5 mL sampling tube (Ina-Optica Co., Ltd), an aqueous solution(0.5 mL) of 11-azide-3,6,9-trioxaundecane-1-amine (0.096 mL, 0.485 mmol)and disialooctasaccharide (50 mg, 0.24 mmol) were added, and theresultant was stirred for 1 hour and then freeze-dried. Into the 5 mLsampling tube after freeze-drying, an N,N-dimethylformamide solution(0.6 mL) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (92 mg, 0.24 mmol) and diisopropylethylamine (0.042mL, 0.24 mmol) were added, followed by stirring at 37° C. for 4 hours.After the completion of the reaction, the reaction solution wastransferred into a centrifuge tube (50 mL) into which diethyl ether (20mL) had been added in advance. The solid matter was precipitated byusing a small centrifuge (Hitachi Koki Co., Ltd., CF16RX) and thesupernatant was removed. Diethyl ether (20 mL) was added and theresultant was decanted. Subsequently, acetonitrile (20 mL) was added andthe resultant was decanted, and then dried under reduced pressure toafford a crude product. The resulting solid matter was dissolved in anappropriate amount of a 0.2% trifluoroacetic acid aqueous solution, andsubjected to separation/purification by reversed-phase HPLC. The eluentwas a 0.1% trifluoroacetic acid aqueous solution and a 0.1%trifluoroacetic acid acetonitrile solution, the apparatus used was aPurif-Rp2 (produced by Shoko Scientific Co., Ltd.), and the column usedwas an Inertsil ODS-3 (produced by GL Sciences, Inc.). Fractionscontaining the desired compound UV-detected (220 nm) during the elutionwere collected together, and freeze-dried to afford the desired compound(42 mg).

Step 2: [N₃-PEG (3)]₂-SG (10)-Ox

Into a 5 mL sampling tube (produced by Ina-Optica Co., Ltd), thecompound synthesized in step 1 (40 mg) and an aqueous solution (200 μL)of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium-chloride (produced byFUSHIMI Pharmaceutical Co., Ltd. 17.9 mg, 0.083 mmol) was added. To thereaction solution after being ice-cooled, an aqueous solution (200 μL)of tripotassium phosphate (52.6 mg, 0.25 mmol) was added, followed bystirring under ice-cooling for 2 hours. The resulting reaction solutionwas subjected to ultrafiltration with an Amicon Ultra (Ultracel 30K,produced by Merck Millipore) to remove the solid matter. The filteredsolution was purified by gel filtration chromatography. The apparatusused was a Purif-Rp2 (produced by Shoko Scientific Co., Ltd.), thecolumn used was a HiPrep 26/10 Desalting (produced by GE Healthcare),the mobile phase used was 0.03%-NH₃ aqueous solution, and the flow ratewas 10 mL/min and the fraction volume was 10 mL. Fractions containingthe desired compound UV-detected (220 nm) during the elution werecollected together, to which a 1 N aqueous solution of sodium hydroxide(33 μL, 0.033 mmol) was added, and the resultant was freeze-dried toafford the desired compound (34 mg).

Example 56: [N3-PEG (3)]-MSG1-Ox

(FIG. 52 , the schematic diagram in the right of the structural formularepresents the corresponding structure in the schematic diagram of anintermediate having a linker structure to which an azide group has beenintroduced as represented by the reaction formula of each of Examples60, 61, 62, 63, 64, 65, and 66.)

Step 1: (MSG1-)Asn

The commercially available product monosialo-Asn free(1S2G/1G2S-10NC-Asn, produced by GlyTech, Inc.) (referred to as“(MSG-)Asn”) (500 mg) was subjected to separation/purification byreversed-phase HPLC under conditions below to separate into (MSG1-)Asneluted as the 1st main peak (retention time: around 15 to 19 min) and(MSG2-)Asn eluted as the 2nd main peak (retention time: around 21 to 26min). The eluent used was a 0.1% formic acid aqueous solution, theapparatus used was an ELS-PDA trigger preparative system (produced byJASCO Corporation), the column used was an Inertsil ODS-3 (10 um,30ϕ×250 mm, produced by GL Sciences, Inc.), and the flow rate was 30mL/min. Fractions belonging to the first peak UV-detected (210 nm)during the elution were collected together, and freeze-dried to affordthe desired compound (238 mg).

Step 2: MSG1

The compound obtained in step 1 (229 mg) was dissolved in 200 mMphosphate buffer solution (pH 6.25) (1145 μL), to which an aqueoussolution (100 μL) of EndoM (produced by Tokyo Chemical Industry Co.,Ltd., 1 U/mL)) was added, and the resultant was incubated at 35° C. for6 days. After the completion of the reaction, the reaction solution wassubjected to ultrafiltration with a VIVASPIN 15R (Hydrosart membrane,30K, 6,000 g), and the filtered solution obtained was subjected toseparation/purification by reversed-phase HPLC. The eluent used was a0.1% trifluoroacetic acid aqueous solution, the apparatus used was anELS-PDA trigger preparative system (produced by JASCO Corporation), andthe column used was an Inertsil ODS-3 (produced by GL Sciences, Inc.).Fractions containing the desired compound UV-detected (210 nm) duringthe elution were collected together, and freeze-dried to afford thedesired compound (117 mg).

Step 3: [N₃-PEG (3)]-MSG1

The compound synthesized in step 2 (169 mg) was reacted in the samemanner as in step 1 of Example 55 to afford the desired compound (94.2mg).

Step 4 [N₃-PEG (3)]-MSG1-Ox

The compound (100 mg) synthesized in step 3 was reacted in the samemanner as in step 2 of Example 55 to afford the desired compound (89mg).

Example 57: [N₃-PEG (3)]-MSG-Ox

(FIG. 53 , the schematic diagram in the right of the structural formularepresents the corresponding structure in the schematic diagram of anintermediate having a linker structure to which an azide group has beenintroduced as represented by the reaction formula of Example 59.)

Step 1: Preparation of (MSG-)Asn

The commercially available product 1S2G/1G2S-10NC-Asn-Fmoc (produced byGlyTech, Inc.) (referred to as “Fmoc-(MSG-)Asn”) (1000 mg) was dissolvedin ethanol/water (1/1) (10 mL), to which a 1 N aqueous solution ofsodium hydroxide (1.75 mL, 4 equivalents) was added, followed bystirring at room temperature for 3 hours. After the completion of thereaction, the reaction solution was subjected to ultrafiltration with anAmicon Ultra (30K, produced by Millipore Corporation) to remove thesolid matter, and 1 N hydrochloric acid (832 μL, 1.9 equivalents) wasadded to the filtered solution obtained. The solvent was removed withthe high-speed evaporator V-10 (produced by Biotage). Acetonitrile wasadded thereto, and the solvent was removed with the high-speedevaporator V-10 (produced by Biotage), and the resultant was thensubjected to separation/purification by reversed-phase HPLC. The eluentwas a 0.1% trifluoroacetic acid aqueous solution and a 0.1%trifluoroacetic acid acetonitrile solution, the apparatus used was aPurif-Rp2 (produced by Shoko Scientific Co., Ltd.), and the column usedwas an Inertsil ODS-3 (produced by GL Sciences, Inc.). Fractionscontaining the desired compound UV-detected (220 nm) during the elutionwere collected together, and freeze-dried. This was dissolved again inpure water, and a pH test paper strip indicated that the solution wasacidic. Hence, 18% aqueous ammonia (150 μL) was added thereto and it wasconfirmed with a pH test paper strip that the solution had become basic,and the solution was freeze-dried again. The desired compound obtaind(840 mg) was directly used for the subsequent reaction.

Step 2: Synthesis of MSG

The compound obtained in step 1 (840 mg) was dissolved in 200 mMphosphate buffer solution (pH 6.25) (6000 μL), to which an aqueoussolution (200 μL) of EndoM (produced by Tokyo Chemical Industry Co.,Ltd., 1 U/mL)) was added, and the resultant was incubated at 28° C. for26 hours. Because the reaction had not completed, an aqueous solution(50 μL) of EndoM (produced by Tokyo Chemical Industry Co., Ltd., 1U/mL)) was added, and the resultant was incubated at 28° C. for 2 hours,and then left to stand at room temperature until the completion of thereaction. After the completion of the reaction, the reaction solutionwas subjected to ultrafiltration with an Amicon Ultra (30K, produced byMillipore Corporation). Trifluoroacetic acid (80 μL) was added to thefiltered solution obtained, which was subjected toseparation/purification by reversed-phase HPLC. The eluent was a 0.1%trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acidacetonitrile solution, the apparatus used was a Purif-Rp2 (produced byShoko Scientific Co., Ltd.), and the column used was an Inertsil ODS-3(produced by GL Sciences, Inc.). Fractions containing the desiredcompound UV-detected (220 nm) during the elution were collectedtogether, and freeze-dried. This was dissolved again in pure water inorder to remove the residual trifluoroacetic acid, and thus the desiredcompound (618 mg) was obtained as a colorless solid.

ESI-MS: Calcd for C₆₆H₁₁₀N₄O₄₉: [M+H]⁺ 1743.62, Found 1743.63

Step 3: Synthesis of [N₃-PEG (3)]-MSG

In accordance with the procedure of step 1 of Example 55 using thecompound obtained in step 2 (120 mg), the desired compound (88.6 mg) wasobtained.

ESI-MS: Calcd for C₇₃H₂₄N₈O₅₁: [M+2H]²⁺ 965.37, Found 965.37

Step 4 Synthesis of [N₃-PEG (3)]-MSG-Ox

In accordance with the procedure of step 2 of Example 55 using thecompound obtained in step 4 (100 mg), the desired compound (88 mg) wasobtained.

Preparation of Glycan-Remodeled Antibody Example 58: Sugar ChainRemodeling 1 (T-SG)

(See FIG. 54 . This formula represents a linker structure in which anazide group has been introduced to a sialic acid at the non-reducingterminal of an SG-type N297 glycan. In Example 58, linker structures ofintermediates formed by introducing an azide group to an N297 glycan areall the same as the structure represented by the formula.)

Step 1: Preparation of (Fucα1,6)GlcNAc-Trastuzumab

The 22 mg/mL trastuzumab solution (25 mM histidine solution (pH 6.0), 5%sorbitol solution) (45.5 mL) prepared in Reference Example 3 was halvedand according to common operation C, buffer exchange to 50 mM phosphatebuffer (pH 6.0) was conducted twice separately. To the resulting 28.1mg/mL (18 mL) and 28.0 mg/mL (18 mL) trastuzumab solution (50 mMphosphate buffer (pH 6.0)), 1.26 mL and 1.27 mL of wild-type EndoSsolution (2.0 mg/mL, PBS) were respectively added, and the solutionswere incubated at 37° C. for 4 hours. The progress of the reaction waschecked by Experion electrophoresis station (produced by Bio-RadLaboratories, Inc.). After the completion of the reaction, purificationby affinity chromatography and purification with a hydroxyapatite columnwere performed in accordance with the following methods.

(1) Purification by Affinity Chromatography

Purification apparatus: AKTA pure150 (produced by GE Healthcare)

Column: HiTrap rProtein A FF (5 mL) (produced by GE Healthcare)

Flow rate: 5 mL/min (1.25 mL/min in charging)

Each reaction solution obtained above was purified in multiple separateoperations. Two columns were linked together into one column, and inconnecting to the column the reaction solution was added to the upperpart of the column, and 2 CV of binding buffer (20 mM phosphate buffer(pH 6.0)) was flowed at 1.25 mL/min and 5 CV thereof was further flowedat 5 mL/min. In intermediate washing, 15 CV of washing solution (20 mMphosphate buffer (pH 7.0), 0.5 M sodium chloride solution) was flowed.In elution, 6 CV of elution buffer (ImmunoPure IgG Eution buffer,produced by Pierce) was flowed. The eluate was immediately neutralizedwith 1 M Tris buffer (pH 9.0). Fractions UV-detected (280 nm) during theelution were checked by using the micro-volume spectrophotometer Xpose(produced by Trinean NV) and an Experion electrophoresis station(produced by Bio-Rad Laboratories, Inc.). Fractions containing thedesired compound were subjected to buffer exchange to 5 mM phosphatebuffer/50 mM 2-morpholinoethanesulfonic acid (MES) solution (pH 6.8) byusing common operation C.

(2) Purification by Hydroxyapatite Chromatography

Purification apparatus: AKTA avant25 (produced by GE Healthcare)

Column: Bio-Scale Mini CHT Type I cartridge (5 mL) (produced by Bio-RadLaboratories, Inc.)

Flow rate: 5 mL/min (1.25 mL/min in charging)

Two columns were linked together into one column, and the solutionobtained in (1) was purified in multiple separate operations. Thesolution was added to the upper part of the column, and 2 CV of solutionA (5 mM phosphate buffer, 50 mM 2-morpholinoethanesulfonic acid (MES)solution (pH 6.8)) was flowed at 1.25 mL/min and 3 CV thereof wasfurther flowed at 5 mL/min. Thereafter, elution was performed withsolution A and solution B (5 mM phosphate buffer/50 mM2-morpholinoethanesulfonic acid (MES) solution (pH 6.8), 2 M sodiumchloride solution). The elution conditions were solution A:solutionB=100:0 to 0:100 (15 CV). Further, 5 CV of washing solution (500 mMphosphate buffer (pH 6.5)) was flowed.

Fractions containing the desired compound were subjected to bufferexchange by using common operation C to afford a 25.5 mg/A D mL(Fucα1,6)GlcNAc-Trastuzumab solution (50 mM phosphate buffer (pH 6.0))(35 mL).

Step 2: Preparation of Trastuzumab[SG-(N₃)₂]₂

To the 23.9 mg/mL (Fucα1,6)GlcNAc-Trastuzumab solution (50 mM phosphatebuffer (pH 6.0)) obtained in step 1 (3.37 mL), a solution (0.258 mL) ofthe compound synthesized in step 2 of Example 55 (12.9 mg) in 50 mMphosphate buffer (pH 6.0) and 4.90 mg/mL EndoS D233Q/Q303L solution(PBS) (0.328 mL) were added, and the resultant was incubated at 30° C.for 4.5 hours. These operations were performed in two lots. The progressof the reaction was checked by using an Experion electrophoresis station(produced by Bio-Rad Laboratories, Inc.). After the completion of thereaction, purification by affinity chromatography and purification byhydroxyapatite chromatography were performed as in step 1, and fractionscontaining the desired compound were then subjected to buffer exchangeto phosphate buffered saline (pH 6.0) by using common operation C toafford a 10.0 mg/mL Trastuzumab [SG-(N₃)₂]₂ solution (phosphate bufferedsaline (pH 6.0)) (15.5 mL).

Example 59: Sugar Chain Remodeling 2 (T-MSG)

(See FIG. 55 . This formula represents a linker structure in which anazide group has been introduced to a sialic acid at the non-reducingterminal of an MSG-type N297 glycan. In Example 59, linker structures ofintermediates formed by introducing an azide group to an N297 glycan areall the same as the structure represented by the formula.)

Step 1: Trastuzumab[MSG-N₃]₂

The following operations were performed in five lots. The compoundobtained in step 1 of Example 58 (20 mg/mL, 15.0 mL) was used togetherwith the compound obtained in step 4 of Example 57 (25.5 mg) as a glycandonor, and incubated at 30° C. for 3 hours, and the operations same asin step 2 of Example 59 were performed. With the five lots combined, a14.4 mg/mL Trastuzumab [MSG-N₃]₂ solution (phosphate buffered saline (pH6.0)) (93.5 mL) was obtained.

Example 60: Sugar Chain Remodeling 3 (T-MSG1)

(See FIG. 56 . This formula represents a linker structure in which anazide group has been introduced to a sialic acid at the non-reducingterminal of an MSG1-type N297 glycan. In Example 60, linker structuresof intermediates formed by introducing an azide group to an N297 glycanare all the same as the structure represented by the formula. The sameholds true for Examples 61 to 66.)

Step 1: Trastuzumab[MSG1-N₃]₂

The following operations were performed in two lots. The compoundobtained in step 1 of Example 58 (25.5 mL, 7.8 mL) was used togetherwith the compound obtained in step 4 of Example 56 (25.5 mg) as a glycandonor, and incubated at 30° C. for 3 hours, and the operations same asin step 2 of Example 59 were performed. With the two lots combined, a10.6 mg/mL Trastuzumab[MSG1-N₃]₂ solution (phosphate buffered saline (pH6.0)) (31 mL) was obtained.

Example 61: Sugar Chain Remodeling 4 (CLDN6-MSG1 (H1L1)) Step 1:(Fucα1,6)GlcNAc-Anti-CLDN6 Antibody (H1L1)

The operations same as in step 1 of Example 58 were performed using aca. 37.7 mg/mL anti-CLDN6 antibody solution (25 mM histidine solution(pH 6.0), 5% sorbitol solution) prepared in Example 136 (2.5 mL) toafford a 19.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1) solution(50 mM phosphate buffer (pH 6.0)) (4.8 mL) (see FIG. 57 ).

Step 2: Anti-CLDN6 Antibody (H1L1)-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the19.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 (H1L1) antibody solution (50 mMphosphate buffer (pH 6.0)) obtained in step 1 (4.8 mL) to afford a 10.2mg/mL anti-CLDN6 antibody (H1L1)-[MSG1-N₃]₂ solution (phosphate bufferedsaline (pH 6.0)) (7.2 mL).

Example 62: Sugar Chain Remodeling 5 (CLDN6-MSG1 (H2L2)) Step 1:(Fucα1,6)GlcNAc-Anti-CLDN6 Antibody (H2L2)

The operations same as in step 1 of Example 58 were performed using aca. 20 mg/mL anti-CLDN6 antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) prepared in Example 136 (6 mL) to afford a21.84 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H2L2) solution (50 mMphosphate buffer (pH 6.0)) (5.7 mL) (see FIG. 58 ).

Step 2: Anti-CLDN6 Antibody (H2L2)-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the21.8 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 (H2L2) antibody solution (50 mMphosphate buffer (pH 6.0)) obtained in step 1 (5.7 mL) to afford a 10.2mg/mL anti-CLDN6 antibody (H2L2)-[MSG1-N₃]₂ solution (phosphate bufferedsaline (pH 6.0)) (11.1 mL).

Example 63: Sugar Chain Remodeling 6 (CLDN6-MSG1 (H1L3)) Step 1:(Fucα1,6)GlcNAc-Anti-CLDN6 Antibody (H1L3)

The operations same as in step 1 of Example 58 were performed using aca. 39.4 mg/mL anti-CLDN6 antibody solution (25 mM histidine solution(pH 6.0), 5% sorbitol solution) prepared in Example 136 (3 mL) to afforda 39.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L3) solution (50 mMphosphate buffer (pH 6.0)) (4.5 mL) (see FIG. 59 ).

Step 2: Anti-CLDN6 Antibody (H1L3)-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the39.2 mg/mL (Fucα1,6)GlcNAc-anti-CLDN6 (H1L3) antibody solution (50 mMphosphate buffer (pH 6.0)) obtained in step 1 (4.5 mL) to afford a 9.83mg/mL anti-CLDN6 antibody (H1L3)-[MSG1-N₃]₂ solution (phosphate bufferedsaline (pH 6.0)) (7.2 mL).

Example 64: Sugar Chain Remodeling 7 (CD98-MSG1) Step 1:(Fucα1,6)GlcNAc-Anti-CD98 Antibody

The operations same as in step 1 of Example 58 were performed using aca. 20 mg/mL anti-CD98 antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) prepared in Reference Example 6 (6 mL) toafford a 21.7 mg/mL (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mMphosphate buffer (pH 6.0)) (4.7 mL) (see FIG. 60 ).

Step 2: Anti-CD98 Antibody-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the21.7 mg/mL (Fucα1,6)GlcNAc-anti-CD98 antibody solution (50 mM phosphatebuffer (pH 6.0)) obtained in step 1 (4.7 mL) to afford a 10.1 mg/mLanti-CD98 antibody-[MSG1-N₃]₂ solution (phosphate buffered saline (pH6.0)) (7.6 mL).

Example 65: Sugar Chain Remodeling 8 (TROP2-MSG1) Step 1:(Fucα1,6)GlcNAc-Anti-Trop2 Antibody

The operations same as in step 1 of Example 58 were performed using aca. 20 mg/mL anti-Trop2 antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) obtained in Reference Example 5 (6 mL) toafford a 21.69 mg/mL (Fucα1,6)GlcNAc-anti-Trop2 antibody solution (50 mMphosphate buffer (pH 6.0)) (3.3 mL) (see FIG. 61 ).

Step 2: Anti-Trop2 Antibody-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the21.69 mg/mL (Fucα1,6)GlcNAc-anti-Trop2 antibody solution (50 mMphosphate buffer (pH 6.0)) obtained in step 1 (3.35 mL) to afford a 10.3mg/mL anti-Trop2 antibody-[MSG1-N₃]₂ solution (phosphate buffered saline(pH 6.0)) (6.4 mL).

Example 66: Sugar Chain Remodeling 9 (LPS-MSG1) Step 1:(Fucα1,6)GlcNAc-Anti-LPS Antibody

The operations same as in step 1 of Example 58 were performed using aca. 17 mg/mL anti-LPS antibody solution (25 mM histidine solution (pH6.0), 5% sorbitol solution) prepared in Reference Example 4 (6.6 mL) toafford a 21.03 mg/mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mMphosphate buffer (pH 6.0)) (5.4 mL) (see FIG. 62 ).

Step 2: Anti-LPS Antibody-[MSG1-N₃]₂

The operations same as in step 1 of Example 60 were performed using the21.03 mg/mL (Fucα1,6)GlcNAc-anti-LPS antibody solution (50 mM phosphatebuffer (pH 6.0)) obtained in step 1 (5.4 mL) to afford a 9.89 mg/mLanti-LPS antibody-[MSG1-N₃]₂ solution (phosphate buffered saline (pH6.0)) (7.9 mL).

[Synthesis of ADC]

ADCs described in Examples 67 to 71, 77 to 80, 82 to 88, 92 to 95, 109to 114, and 120 were synthesized, as illustrated in the followingreaction formula, by conjugating the antibody obtained in step 1 ofExample 59 with a drug-linker. In the formula, R differs amongdrug-linkers used in those Examples (see FIG. 63 ).

ADCs described in Examples 72, 73, 75, and 91 were synthesized, asillustrated in the following reaction formula, by conjugating theantibody obtained in step 2 of Example 58 with a drug-linker. In theformula, R differs among drug-linkers used in those Examples (see FIG.64 ).

ADCs described in Examples 74, 81, 89, 90, 96 to 105, 115, and 118 weresynthesized, as illustrated in the following reaction formula, byconjugating the antibody obtained in step 1 of Example 60 with adrug-linker. In the formula, R group differs among drug-linkers used inthose Examples (see FIG. 65 ).

Example 67: ADC1

The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 67 has a linker as a mixture ofthe two structures shown as R (FIG. 66 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 24 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 14.00 mL of a solution of the desired compound.This solution was concentrated by using common operation A to afford0.75 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 11.4 mg/mL, antibody yield: 8.56 mg (86%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 68: ADC2

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 68 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 14 of Example 25 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.48 mg/mL, antibody yield: 8.88 mg (89%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 69: ADC3

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 69 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 14 of Example 26 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.45 mg/mL, antibody yield: 8.67 mg (89%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 70: ADC4

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 70 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 27 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.30 mg/mL, antibody yield: 7.80 mg (78%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 71: ADC5

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 71 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 28 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.08 mg/mL, antibody yield: 6.48 mg (65%),average number of conjugated drug molecules per antibody molecule (n):1.6

Example 72: ADC6

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 72 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 2 of Example 58, 1,2-propanediol (0.835mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 27 (0.165 mL; 24 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 4.50 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.75 mg/mL, antibody yield: 7.86 mg (79%),average number of conjugated drug molecules per antibody molecule (n):3.8

Example 73: ADC7

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 73 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 2 of Example 58, 1,2-propanediol (0.835mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 4 (0.165 mL; 24 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 4.50 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.66 mg/mL, antibody yield: 7.48 mg (75%),average number of conjugated drug molecules per antibody molecule (n):3.8

Example 74: ADC8

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 74 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 14 of Example 29 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.36 mg/mL, antibody yield: 8.17 mg (82%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 75: ADC9

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 75 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 2 of Example 58, 1,2-propanediol (0.835mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 9 of Example 9 (0.165 mL; 24 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 8.50 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.86 mg/mL, antibody yield: 7.35 mg (73%),average number of conjugated drug molecules per antibody molecule (n):3.6

Example 76: ADC10

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 76 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

As illustrated in the reaction formula in Example 106, to a phosphatebuffered saline (pH 6.0) solution of the antibody (10.2 mg/mL, 0.400 mL)obtained in step 2 of Example 61, 1,2-propanediol (0.200 mL), a 10 mMdimethyl sulfoxide solution of the compound obtained in step 2 ofExample 30 (0.0549 mL; 20 equivalents per antibody molecule), anddimethyl sulfoxide (0.145 mL) were added at room temperature, and theresultant was reacted using a tube rotator (MTR-103, AS ONE Corporation)at room temperature for 72 hours.

Purification operation: The solution was purified by using commonoperation D to afford 2.50 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.19 mg/mL, antibody yield: 2.98 mg (75%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 77: ADC11

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 77 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.88mg/mL, 0.500 mL) obtained in step 1 of Example 59, 1,2-propanediol(0.459 mL) and a 10 mM dimethyl sulfoxide solution of the compoundobtained in step 12 of Example 19 (0.0408 mL; 12 equivalents perantibody molecule) were added at room temperature, and the resultant wasreacted using a tube rotator (MTR-103, AS ONE Corporation) at roomtemperature for 48 hours.

Purification operation: The solution was purified by using commonoperation D, and the resulting solution was concentrated by using commonoperation A to afford 0.470 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 8.31 mg/mL, antibody yield: 3.90 mg (79%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 78: ADC12

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 78 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 20 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D, and the resulting solution was then concentrated by usingcommon operation A to afford 0.75 mL of a solution of the desiredcompound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 8.70 mg/mL, antibody yield: 6.94 mg (69%),average number of conjugated drug molecules per antibody molecule (n):1.7

Example 79: ADC13

The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 79 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 18 of Example 21 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.96 mg/mL, antibody yield: 5.77 mg (58%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 80: ADC14

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 80 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 22 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.28 mg/mL, antibody yield: 7.70 mg (77%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 81: ADC15

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 81 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 10 of Example 23 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.60 mg/mL, antibody yield: 9.60 mg (96%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 82: ADC16

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 82 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.88mg/mL, 0.500 mL) obtained in step 1 of Example 59, 1,2-propanediol(0.459 mL) and a 10 mM dimethyl sulfoxide solution of the compoundobtained in step 11 of Example 34 (0.0408 mL; 12 equivalents perantibody molecule) were added at room temperature, and the resultant wasreacted using a tube rotator (MTR-103, AS ONE Corporation) at roomtemperature for 1 day.

Purification operation: The solution was purified by using commonoperation D to afford 10.5 mL of a solution of the desired compound.This solution was concentrated by using common operation A to afford0.500 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 6.94 mg/mL, antibody yield: 3.47 mg (70%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 83: ADC17

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 83 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.88mg/mL, 0.500 mL) obtained in step 1 of Example 59, 1,2-propanediol(0.459 mL) and a 10 mM dimethyl sulfoxide solution of the compoundobtained in step 4 of Example 35 (0.0408 mL; 12 equivalents per antibodymolecule) were added at room temperature, and the resultant was reactedusing a tube rotator (MTR-103, AS ONE Corporation) at room temperaturefor 1 day.

Purification operation: The solution was purified by using commonoperation D, and the resulting solution was concentrated by using commonoperation A to afford 0.500 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 8.07 mg/mL, antibody yield: 4.03 mg (82%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 84: ADC18

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 86 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 7 of Example 36 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 1day.

Purification operation: The solution was purified by using commonoperation D to afford 14.0 mL of a solution of the desired compound.This solution was concentrated by using common operation A to afford0.700 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 12.9 mg/mL, antibody yield: 9.00 mg (90%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 85: ADC19

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 85 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 37 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.33 mg/mL, antibody yield: 7.97 mg (80%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 86: ADC20

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 86 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 38 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.56 mg/mL, antibody yield: 9.37 mg (94%),average number of conjugated drug molecules per antibody molecule (n):2.0

Example 87: ADC21

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 87 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 6 of Example 39 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.89 mg/mL, antibody yield: 11.4 mg(quantitative), average number of conjugated drug molecules per antibodymolecule (n): 1.9

Example 88: ADC22

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in Example 88 has a linker as a mixture of the twostructures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 6 of Example 40 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.84 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 89: ADC23

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 89 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 37 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.46 mg/mL, antibody yield: 8.76 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 90: ADC24

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 90 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 2 of Example 41 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.53 mg/mL, antibody yield: 9.17 mg (92%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 91: ADC25

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in Example 91 has a linker as a mixture of the twostructures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 2 of Example 58, 1,2-propanediol (0.835mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 37 (0.165 mL; 24 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 10.5 mL of a solution of the desired compound.Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.73 mg/mL, antibody yield: 7.70 mg (77%),average number of conjugated drug molecules per antibody molecule (n):3.8

Example 92: ADC26

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 92 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.88mg/mL, 0.500 mL) obtained in step 1 of Example 59, 1,2-propanediol(0.459 mL) and a 10 mM dimethyl sulfoxide solution of the compoundobtained in step 10 of Example 15 (0.0408 mL; 12 equivalents perantibody molecule) were added at room temperature, and the resultant wasreacted using a tube rotator (MTR-103, AS ONE Corporation) at roomtemperature for 1 day.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.This solution was concentrated by using common operation A to afford0.420 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 7.27 mg/mL, antibody yield: 3.54 mg (72%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 93: ADC27

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 93 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.88mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 16 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 1day.

Purification operation: The solution was purified by using commonoperation D to afford 10.5 mL of a solution of the desired compound.This solution was concentrated by using common operation A to afford0.850 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 8.90 mg/mL, antibody yield: 7.56 mg (77%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 94: ADC28

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 94 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 17 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.24 mg/mL, antibody yield: 7.43 mg (73%),average number of conjugated drug molecules per antibody molecule (n):1.5

Example 95: ADC29

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 95 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 18 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.29 mg/mL, antibody yield: 7.76 mg (76%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 96: ADC30

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 96 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 5.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (4.59mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 4 (0.413 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 30.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.30 mg/mL, antibody yield: 38.9 mg (78%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 97: ADC31

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 97 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 11 of Example 5 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.82 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 98: ADC32

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 98 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 6 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.35 mg/mL, antibody yield: 8.10 mg (81%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 99: ADC33

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 99 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 10 of Example 7 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.41 mg/mL, antibody yield: 8.44 mg (84%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 100: ADC34

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 100 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 9 of Example 9 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.79 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 101: ADC35

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 101 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 14 of Example 10 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.67 mg/mL, antibody yield: 10.0 mg(quantitative), average number of conjugated drug molecules per antibodymolecule (n): 1.8

Example 102: ADC36

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 103 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 11 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.40 mg/mL, antibody yield: 8.39 mg (84%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 103: ADC37

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 103 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 14 of Example 12 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.41 mg/mL, antibody yield: 8.49 mg (85%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 104: ADC: 38

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 104 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 2 of Example 13 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.63 mg/mL, antibody yield: 9.80 mg (98%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 105: ADC39

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 105 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 1 of Example 14 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.72 mg/mL, antibody yield: 10.3 mg(quantitative), average number of conjugated drug molecules per antibodymolecule (n): 1.9

Example 106: ADC40

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 106 has a linker as a mixture ofthe two structures shown as R) (see FIG. 67 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 2.50 mL) obtained in step 2 of Example 61, 1,2-propanediol (2.29mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.206 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 14.5 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.54 mg/mL, antibody yield: 22.3 mg (89%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 107: ADC41

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 107 has a linker as a mixture ofthe two structures shown as R) (see FIG. 68 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.96mg/mL, 2.50 mL) obtained in step 2 of Example 62, 1,2-propanediol (2.29mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.206 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 14.5 mL of a solution of the desired compound.Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.52 mg/mL, antibody yield: 22.0 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 108: ADC42

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 108 has a linker as a mixture ofthe two structures shown as R) (see FIG. 69 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.83mg/mL, 2.50 mL) obtained in step 2 of Example 63, 1,2-propanediol (2.29mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.206 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 14.5 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.45 mg/mL, antibody yield: 21.0 mg (84%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 109: ADC43

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 109 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 16 of Example 42 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 3.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.30 mg/mL, antibody yield: 7.79 mg (78%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 110: ADC44

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 110 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 43 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.93 mg/mL, antibody yield: 5.58 mg (56%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 111: ADC45

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 111 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 44 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6.0 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.99 mg/mL, antibody yield: 5.95 mg (59%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 112: ADC46

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 112 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 31 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.49 mg/mL, antibody yield: 8.94 mg (89%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 113: ADC47

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 113 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 32 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.45 mg/mL, antibody yield: 8.73 mg (87%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 114: ADC48

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 114 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 8 of Example 33 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.45 mg/mL, antibody yield: 8.70 mg (87%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 115: ADC49

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 115 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.41 mg/mL, antibody yield: 8.45 mg (85%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 116: ADC50

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 116 has a linker as a mixture ofthe two structures shown as R) (see FIG. 70 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10mg/mL, 1.00 mL) obtained in step 2 of Example 65, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.8 mg (88%),average number of conjugated drug molecules per antibody molecule (n):1.9

Example 117: ADC51

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 117 has a linker as a mixture ofthe two structures shown as R) (see FIG. 71 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.2mg/mL, 1.00 mL) obtained in step 2 of Example 64, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.36 mg/mL, antibody yield: 8.19 mg (82%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 118: ADC52

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 118 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 60, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 2 of Example 8 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 3days.

Purification operation: The solution was purified by using commonoperation D to afford 6.00 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.06 mg/mL, antibody yield: 6.35 mg (63%),average number of conjugated drug molecules per antibody molecule (n):1.2

Example 119: ADC53

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 119 has a linker as a mixture ofthe two structures shown as R) (see FIG. 72 ).

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (9.89mg/mL, 0.40 mL) obtained in step 2 of Example 66, 1,2-propanediol (0.367mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 13 of Example 3 (0.0328 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 2days.

Purification operation: The solution was purified by using commonoperation D to afford 2.50 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.16 mg/mL, antibody yield: 2.89 mg (72%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 120: ADC54

(The triazole ring to be formed in step 1 has geometric isomers, and thecompound obtained in step 1 of Example 120 has a linker as a mixture ofthe two structures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a phosphate buffered saline (pH 6.0) solution of the antibody (10.0mg/mL, 1.00 mL) obtained in step 1 of Example 59, 1,2-propanediol (0.917mL) and a 10 mM dimethyl sulfoxide solution of the compound obtained instep 12 of Example 4 (0.0825 mL; 12 equivalents per antibody molecule)were added at room temperature, and the resultant was reacted using atube rotator (MTR-103, AS ONE Corporation) at room temperature for 48hours.

Purification operation: The solution was purified by using commonoperation D to afford 6 mL of a solution of the desired compound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 1.49 mg/mL, antibody yield: 8.94 mg (89%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 121: Anticellular Effect of Antibody-Drug Conjugate (1)

NCI-N87 (American Type Culture Collection; ATCC CRL-5822), a humangastric cancer cell line of HER2 antigen-positive cells, was cultured inRPMI1640 Medium (Thermo Fisher Scientific; hereinafter, referred to asRPMI medium) containing 10% fetal bovine serum (Hyclone). MDA-MB-468(ATCC HTB-132), HER2 antigen-negative cells, was cultured in Leibovitz'sL-15 Medium (Thermo Fisher Scientific; hereinafter, referred to asLeibovitz's medium) containing 10% fetal bovine serum (Hyclone). NCI-N87and MDA-MB-468 cells were prepared with RPMI medium and Leibovitz'smedium, respectively, to reach 2.5×10⁴ cells/mL, and 80 μL portions ofthem were added to a 96-well cell culture microplate. After addition ofthe cells, NCI-N87 was cultured at 37° C. and 5% CO₂ overnight, andMDA-MB-468 was cultured at 37° C. overnight, without setting of CO₂concentration.

On the next day, 20 μL portions of an anti-HER2 antibody-drug conjugatediluted with RPMI medium or Leibovitz's medium to 100 nM, 10 nM, 1 nM,0.1 nM, 0.01 nM, and 0.001 nM were added to the microplate. To each wellwithout any antibody-drug conjugate, 20 μL of RPMI medium or Leibovitz'smedium was added. NCI-N87 was cultured at 37° C. and 5% CO₂ for 6 days,and MDA-MB-468 was cultured at 37° C. for 6 days, without setting of CO₂concentration. After culturing, the microplate was taken out of theincubator, and left to stand at room temperature for 30 minutes.CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation) inan amount equivalent to that of the culture solution was added, andstirred using a plate mixer. The microplate was left to stand at roomtemperature for 10 minutes, and thereafter the amount of emission wasmeasured by using a plate reader (PerkinElmer).

Cell survival rates were calculated by using the following formula.Cell survival rate (%)=a+b×100a: Mean value of amounts of emission from wells with test substanceb: Mean value of amounts of emission from wells with mediumIC₅₀ values were calculated by using the following formula.IC₅₀ (nM)=antilog((50−d)×(LOG₁₀(b)−LOG₁₀(a))+(d−c)+LOG₁₀(b))a: Concentration of test substance, ab: Concentration of test substance, bc: Cell survival rate when test substance of concentration a was addedd: Cell survival rate when test substance of concentration b was addeda and b satisfy a>b at points sandwiching a cell survival rate of 50%.

The antibody-drug conjugates ADC17, ADC18, ADC1, ADC54, ADC20, ADC34,ADC23, ADC24, and ADC25 each exhibited an anticellular effect ofIC₅₀<0.001 (nM) on the NCI-N87 cells. The antibody-drug conjugatesADC26, ADC27, ADC16, ADC11, ADC12, ADC2, ADC3, ADC4, ADC43, ADC5, ADC21,ADC48, ADC44, ADC6, ADC7, ADC28, ADC29, ADC13, ADC14, ADC30, ADC31,ADC32, ADC33, ADC35, ADC36, ADC8, ADC9, ADC38,

and ADC39 each exhibited an anticellular effect of 0.001≤IC₅₀<0.1 (nM).None of the antibody-drug conjugates exhibited anticellular effect onthe MDA-MB-468 cells (IC₅₀>0.1 (nM)).

Example 122: Anticellular Effect of Antibody-Drug Conjugate (2)

NCI-N87 (American Type Culture Collection; ATCC CRL-5822), a humangastric cancer cell line of HER2 antigen-positive cells, was cultured inRPMI1640 Medium (Thermo Fisher Scientific; hereinafter, referred to asRPMI medium) containing 10% fetal bovine serum (Hyclone). JIMT-1(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ ACC589), a human breast cancer cell line of HER2 antigen-positive cells,was cultured in Dulbecco's Modified Eagle Medium (Thermo FisherScientific; hereinafter, referred to as DMEM medium) containing 10%fetal bovine serum (Hyclone). NCI-N87 cells were prepared with RPMImedium to reach 5.0×10⁴ cells/mL and JIMT-1 cells were prepared withDMEM medium to reach 1.3×10⁴ cells/mL, and 80 μL portions of them wereadded to a 96-well cell culture microplate, and the cells were culturedat 37° C. and 5% CO₂ overnight.

On the next day, 20 μL portions of the anti-HER2 antibody-drug conjugateADC49 or anti-LPS antibody-drug conjugate ADC53 diluted with RPMI mediumor DMEM medium to 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.13 nM, 0.026nM, 0.0051 nM, and 0.0010 nM were added to the microplate. To each wellwithout any antibody-drug conjugate, 20 μL of RPMI medium or DMEM mediumwas added. The cells were cultured at 37° C. and 5% CO₂ for 6 days.After culturing, the microplate was taken out of the incubator, and leftto stand at room temperature for 30 minutes. CellTiter-Glo LuminescentCell Viability Assay (Promega Corporation) in an amount equivalent tothat of the culture solution was added, and stirred using a plate mixer.The microplate was left to stand at room temperature for 10 minutes, andthereafter the amount of emission was measured by using a plate reader(PerkinElmer). Cell survival rates were calculated by using thefollowing formula.

Cell survival rate (%)=a+b×100

a: Mean value of amounts of emission from wells with test substance

b: Mean value of amounts of emission from wells with medium

IC₅₀ values were calculated by using the following formula.IC₅₀ (nM)=antilog((50−d)×(LOG₁₀(b)−LOG₁₀(a))+(d−c)+LOG₁₀(b))a: Concentration of test substance, ab: Concentration of test substance, bc: Cell survival rate when test substance of concentration a was addedd: Cell survival rate when test substance of concentration b was addeda and b satisfy a>b at points sandwiching a cell survival rate of 50%.

The anti-HER2 antibody-drug conjugate ADC49 exhibited an anticellulareffect of 0.001<IC50<0.1 (nM) on both of the NCI-N87 and JIMT-1 cells.The anti-LPS antibody-drug conjugate ADC53 exhibited, by contrast, noanticellular effect on both cells (IC₅₀>0.1 (nM)).

Example 123: Anticellular Effect of Antibody-Drug Conjugate (3)

OV-90 (ATCC CRL-11732), a human ovarian cancer cell line of CLDN6antigen-positive cells, was cultured in 1:1 mixed medium (hereinafter,referred to as the medium) of Medium199 (Thermo Fisher Scientific) andMCDB 105 Medium (Sigma-Aldrich Co. LLC) containing 15% fetal bovineserum (Hyclone). OV-90 cells were prepared with the medium to reach1.9×10⁴ cells/mL, and 80 μL portions of them were added to a 96-wellcell culture microplate, and the cells were cultured at 37° C. and 5%CO₂ overnight.

On the next day, 20 μL portions of the anti-CLDN6 antibody-drugconjugate ADC40, ADC41, or ADC42 diluted with the medium to 50 nM, 10nM, 2.0 nM, 400 pM, 80 pM, 16 pM, 3.2 pM, 0.64 pM, and 0.13 pM wereadded to the microplate. To each well without any antibody-drugconjugate, 20 μL of the medium was added. The cells were cultured at 37°C. and 5% CO₂ for 6 days. After culturing, the microplate was taken outof the incubator, and left to stand at room temperature for 30 minutes.CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation) inan amount equivalent to that of the culture solution was added, andstirred using a plate mixer. The microplate was left to stand at roomtemperature for 10 minutes, and thereafter the amount of emission wasmeasured by using a plate reader (PerkinElmer). Cell survival rates werecalculated by using the following formula.Cell survival rate (%)=a+b×100a: Mean value of amounts of emission from wells with test substanceb: Mean value of amounts of emission from wells with mediumIC₅₀ values were calculated by using the following formula.IC₅₀ (nM)=antilog((50−d)×(LOG₁₀(b)−LOG₁₀(a))+(d−c)+LOG₁₀(b))a: Concentration of test substance, ab: Concentration of test substance, bc: Cell survival rate when test substance of concentration a was addedd: Cell survival rate when test substance of concentration b was addeda and b satisfy a>b at points sandwiching a cell survival rate of 50%.

The anti-CLDN6 antibody-drug conjugates ADC40, ADC41, and ADC42 eachexhibited an anticellular effect of 0.001<IC₅₀<0.1 (nM) on the OV-90cells.

Example 124: Antitumor Test for Antibody-Drug Conjugate (1)

Mouse: Four- to five-week-old female BALB/c nude mice (Charles RiverLaboratories Japan, Inc.) were habituated under SPF conditions for 4 to7 days before being used for experiment. To the mice, sterilized pellets(FR-2, Funabashi Farms Co., Ltd.) were fed and sterilized tap water(prepared by adding 5 to 15 ppm sodium hypochlorite solution) wasprovided.

Assay and calculation formula: In all of the studies, the major axis andminor axis of a tumor were measured twice or three times a week by usingan electronic digital caliper (CD-15CX, Mitutoyo Corp.), and the tumorvolume (mm³) was calculated. The calculation formula is as shown below.Tumor volume (mm³)=Major axis (mm)×[Minor axis (mm)]²×½

Each of the antibody-drug conjugates and antibodies was diluted with 10mM acetate buffer, 5% sorbitol, pH 5.5 (NACALAI TESQUE, INC.; ABSbuffer), and a liquid volume of 10 mL/kg was administered into the tailvein. As a control group (Vehicle group), ABS buffer was administered inthe same manner.

NCI-N87 cells (ATCC CRL-5822) were suspended in physiological saline(Otsuka Pharmaceutical Factory, Inc.), and 1×10⁷ cells weresubcutaneously transplanted to the right flank of each female nude mouse(Day 0), and the mice were randomly grouped on Day 7. The anti-HER2antibody-drug conjugate ADC26, ADC19, or ADC54 was administered into thetail vein on Day 7 at a dose of 0.3 mg/kg for ADC26 and at a dose of 1mg/kg for ADC19 and ADC54. As a control group (Vehicle group), ABSbuffer was administered in the same manner.

FIG. 4 shows the results. The anti-HER2 antibody-drug conjugates ADC26,ADC19, and ADC54 were found to have strong antitumor effect causingregression of tumor. No weight loss was found for any of the mice withadministration of any of the anti-HER2 antibody-drug conjugates.

In the following evaluation examples relating to antitumor test, themethod used in the present evaluation example was conducted, unlessotherwise stated.

Example 125: Antitumor Test for Antibody-Drug Conjugate (2)

NCI-N87 cells (ATCC CRL-5822) were suspended in Dulbecco's phosphatebuffered saline (Sigma-Aldrich Co. LLC), and 1×10⁷ cells weresubcutaneously transplanted to the right flank of each female nude mouse(Day 0), and the mice were randomly grouped on Day 4. The anti-HER2antibody-drug conjugate ADC49, the anti-HER2 antibody trastuzumab(Reference Example 3), or the anti-LPS antibody-drug conjugate ADC53 wasadministered into the tail vein on Day 4 at a dose of 0.33 mg/kg for allcases. As a control group (Vehicle group), ABS buffer was administeredin the same manner.

FIG. 5 shows the results. The anti-HER2 antibody-drug conjugate ADC49was found to have strong antitumor effect causing regression of tumor.By contrast, the anti-HER2 antibody trastuzumab and the anti-LPSantibody-drug conjugate ADC53 did not suppress tumor growth. No weightloss caused by administration of the antibody-drug conjugate ADC49 orADC53, or the anti-HER2 antibody was found for the mice.

Example 126: Antitumor Test for Antibody-Drug Conjugate (3)

KPL-4 cells (Dr. Junichi Kurebayashi, Kawasaki Medical School, BritishJournal of Cancer, (1999) 79 (5/6). 707-717) were suspended inDulbecco's phosphate buffered saline (Sigma-Aldrich Co. LLC), and1.5×10⁷ cells were subcutaneously transplanted to the right flank ofeach female nude mouse (Day 0), and the mice were randomly grouped onDay 14. The anti-HER2 antibody-drug conjugate ADC49, the anti-LPSantibody-drug conjugate ADC53, or trastuzumab tesirine (ReferenceExample 1) was administered into the tail vein on Day 14 at a dose of0.4 mg/kg. As a control group (Vehicle group), ABS buffer wasadministered in the same manner.

FIG. 6 shows the results. The anti-HER2 antibody-drug conjugate ADC49was found to have strong antitumor effect causing regression of tumor.By contrast, regression of tumor was not found for ADC53 and trastuzumabtesirine. No weight loss caused by administration of ADC49 ortrastuzumab tesirine was found for the mice.

Example 127: Antitumor Test for Antibody-Drug Conjugate (4)

JIMT-1 cells (DSMZ ACC 589) were suspended in physiological saline(Otsuka Pharmaceutical Factory, Inc.), and 5×10⁶ cells weresubcutaneously transplanted to the right flank of each female nude mouse(Day 0), and the mice were randomly grouped on Day 10. The anti-HER2antibody-drug conjugate ADC49 or trastuzumab tesirine (ReferenceExample 1) was administered into the tail vein on Day 10 at a dose of0.4 mg/kg. As a control group (Vehicle group), ABS buffer wasadministered in the same manner.

FIG. 7 shows the results. The anti-HER2 antibody-drug conjugate ADC49was found to have strong antitumor effect causing regression of tumor.For trastuzumab tesirine, by contrast, antitumor effect was found butregression of tumor was not found. No weight loss caused byadministration of ADC49 or trastuzumab tesirine was found for the mice.

Example 128: Antitumor Test for Antibody-Drug Conjugate (5)

OV-90 cells (ATCC CRL-11732) were suspended in Matrigel (CorningIncorporated), and 2.5×10⁶ cells were subcutaneously transplanted to theright flank of each female nude mouse (Day 0), and the mice wererandomly grouped on Day 15. The anti-CLDN6 antibody-drug conjugate ADC40or anti-CLDN6 antibody (H1L1) tesirine was administered into the tailvein on Day 15 at a dose of 0.33 mg/kg. As a control group (Vehiclegroup), ABS buffer was administered in the same manner.

FIG. 8 shows the results. The anti-CLDN6 antibody-drug conjugate ADC40was found to have strong antitumor effect causing regression of tumor.By contrast, regression of tumor was not found for the anti-CLDN6antibody (H1L1)-tesirine. No weight loss caused by administration ofADC40 or H1L1-tesirine was found for the mice.

Example 129: Antitumor Test for Antibody-Drug Conjugate (6)

NIH:OVCAR-3 cells (ATCC HTB-161) were suspended in Matrigel (CorningIncorporated), and 1×10⁷ cells were subcutaneously transplanted to theright flank of each female nude mouse (Day 0), and the mice wererandomly grouped on Day 25. The anti-CLDN6 antibody-drug conjugate ADC40or anti-CLDN6 antibody (H1L1)-tesirine was administered into the tailvein on Day 25 at a dose of 0.33 mg/kg. As a control group (Vehiclegroup), ABS buffer was administered in the same manner.

FIG. 9 shows the results. The anti-CLDN6 antibody-drug conjugate ADC40was found to have strong antitumor effect causing regression of tumor.For H1L1-tesirine, by contrast, antitumor effect was found butregression of tumor was not found. No weight loss caused byadministration of ADC40 or H1L1-tesirine was found for the mice.

Example 130: Antitumor Test for Antibody-Drug Conjugate (7)

FaDu cells (ATCC HTB-43) were suspended in physiological saline (OtsukaPharmaceutical Factory, Inc.), and 3×10⁶ cells were subcutaneouslytransplanted to the right flank of each female nude mouse (Day 0), andthe mice were randomly grouped on Day 10. The anti-TROP2 antibody-drugconjugate ADC50 or the anti-LPS antibody-drug conjugate ADC53 wasadministered into the tail vein on Day 10 at a dose of 0.4 mg/kg. As acontrol group (Vehicle group), ABS buffer was administered in the samemanner.

FIG. 10 shows the results. The anti-TROP2 antibody-drug conjugate ADC50was found to have strong antitumor effect causing regression of tumor.By contrast, the anti-LPS antibody-drug conjugate ADC53 did not suppresstumor growth. No weight loss caused by administration of ADC50 or ADC53was found for the mice.

Example 131: Mouse Anti-CLDN6 Antibody B1-Producing Hybridoma (218B1)and Mouse Anti-CLDN6 Antibody C7-Producing Hybridoma (218C7)

131-1. Immunization of Mice and Acquisition of Hybridomas

1-1) Preparation of Cells for Immunization of Mice

In RPMI-1640 (Roswell Park Memorial Institute-1640) 10% FBS (fetalbovine serum) (+) medium (10 mL or 20 mL), 2×10⁶ or 5×10⁶ NOR-P1 cells(human pancreatic cancer cell line, RIKEN RCB-2139) were cultured for 5days and then collected, and washed twice with PBS (phosphate bufferedsaline) and resuspended in PBS (300 μL).

1-2) Immunization of Mice

Each BALB/c mouse (12-week-old) was intraperitoneally immunized withNOR-P1 cells (2×10⁶ cells) at intervals of about 1 week for the first tofifth immunization. About 2 weeks after the fifth immunization, eachBALB/c mouse was intraperitoneally immunized with NOR-P1 cells (5×10⁶cells). About 3 weeks after the sixth immunization, each BALB/c mousewas intraperitoneally immunized with NOR-P1 cells (2×10⁶ cells). EachBALB/c mouse was intraperitoneally immunized with 2×10⁶ NOR-P1 cells atintervals of about 2 weeks for the 8th to 10th immunization. About 3weeks after the 10th immunization (11th immunization) and 3 daysthereafter (12th immunization, final immunization), each BALB/c mousewas intraperitoneally immunized with 5×10⁶ NOR-P1 cells. Splenocyteswere isolated 3 days after the final immunization.

1-3) Preparation of Splenocytes from Immunized Mice

The spleen was isolated from each immunized mouse, triturated, andsuspended in RPMI1640 10% FBS (+) medium. The cell suspension was passedthrough a Cell Strainer (70 μm, BD Falcon), and then centrifuged at 1500rpm at room temperature for 5 minutes to discard the supernatant.Tris-NH₄Cl solution (20 mM Tris-HCl pH 7.2, 77.6 mM NH₄Cl; 20 mL) wasadded thereto, and the resultant was treated at room temperature for 5minutes. PBS (20 mL) was added thereto, and the resultant wascentrifuged at 1500 rpm at room temperature for 5 minutes. After thesupernatant was discard, RPMI1640 FBS (+) medium (10 mL) was added tothe residue.

1-4) Preparation of Myeloma Cells

P3U1 cells (mouse myeloma cell line) was cultured in RPMI1640 FBS (+)medium for 5 days, and then collected and resuspended in RPMI1640 FBS(+) medium (20 mL).

1-5) Cell Fusion

Splenocytes and myeloma cells were mixed together at 5:1, andcentrifuged at 1500 rpm at room temperature for 5 minutes. The cellswere washed twice with RPMI1640 FBS (−) medium (10 mL), and thencentrifuged (1500 rpm, 5 minutes). The group of cells in theprecipitated fraction obtained was sufficiently loosened, andpolyethylene glycol-1500 (PEG-1500; 1 mL) was then gradually addedthereto with stirring over about 1 minute. After stirring for 3 minutes30 seconds, the resultant was left to stand at room temperature for 30seconds. Thereafter, RPMI medium 10% Low IgG FBS (+) (10 mL) was addedto the cell solution over 1 minute. The cell suspension was centrifuged(1500 rpm, 5 minutes), and the cells in the precipitated fractionobtained were gently loosened, and then gently suspended in HAT medium(RPMI1640 medium containing 10% Low IgG FBS, HAT Media Supplement, and5% BriClone; 200 mL). The suspension was aliquoted into a 96-wellculture plate at 200 μL/well, and cultured in an incubator at 37° C. and5% CO₂ for 6 days.

1-6) Screening of Hybridomas/Preparation of Probe

DT3C, a recombinant complex protein, was produced for the purpose ofassaying internalization of antibodies and immunotoxin activity. ThisDT3C is a protein formed by fusing the catalytic domain of diphtheriatoxin (DT) and the antibody-binding domain of streptococcal protein Gthrough genetic engineering. DT3C specifically binds to the Fc region ofantibodies, and induce cell death through protein synthesis inhibitionwhen being incorporated in a cell. Use of this system allowssimultaneous observation of the internalization of an antibody and thecytocidal effect of immunotoxin (Yamaguchi, M. et al., Biochemical andBiophysical Research Communications 454 (2014) 600-603).

1-7) Screening of Hybridomas with DT3C

To a 96-well plate, 4 μg/mL DT3C (25 μL) was added, and the culturesupernatant of the hybridoma obtained in step 1-5 (25 μL) was furtheradded, and the resultant was incubated at room temperature for 30minutes. NOR-P1 cells (50 μL) were seeded at 2×10⁵ cells/mL (RPMI medium10% Low IgG FBS (+)), and cultured in a CO2 incubator at 37° C. for 3days. Through microscopic observation after culturing, wells with thenumber of adhering cells being about 25% or less of that in using anegative control antibody were determined to be positive. Selectedclones were subjected to one or two subcloning steps to establish eightmonoclonal hybridoma cell lines.

131-2: Identification of Antigen to which Antibody Produced by HybridomaBinds

Antigens were identified for two clones, 218B1 and 218C7, of antibodiesproduced by the hybridomas prepared in Example 131-1.

2-1) Immunoprecipitation of Biotin-Labeled Cell Surface Protein with218B1 Antibody and 218C7 Antibody

Culture supernatant of 2×10⁶ NTERA-2 cells (human testicular cancer cellline, ATCC CRL-1973) was removed, and the residue was washed twice withPBS. EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific) was suspendedin PBS to a concentration of 0.1 mg/mL. After PBS was removed,Biotin/PBS solution was added, and the resultant was incubated on ashaker for 30 minutes, and then washed twice with 100 mM glycine/PBSsolution (25 mL) and then washed once with PBS (10 mL). The washed cellswere resuspended in 200 μL of lysis buffer (150 mM NaCl, 50 mM Tris-HClpH 7.4, 1% DDM, Protease inhibitor, Complete EDTA free (F. Hoffmann-LaRoche, Ltd.) 1 particle/50 mL), and treated at 4° C. for 30 minutes. Theresultant was centrifuged (13000 rpm, 20 minutes, 4° C.) to prepare acell lysate. To the cell lysate, Protein G Sepharose/lysis buffer (50%slurry; 30 μL) obtained by substituting the buffer of Protein GSepharose (Protein G Sepharose 4 Fast Flow (GE Healthcare)) with thelysis buffer was added, and the resultant was rotated at 4° C. for 30minutes and then centrifuged at 4° C. for 1 minute, and the supernatantwas collected. To this supernatant the 218B1 antibody or 218C7 antibody(about 3 μg) was added, and the resultant was rotated at 4° C. for 30minutes, to which Protein G Sepharose/lysis buffer (50% slurry; 60 μL)was then added, and the resultant was rotated at 4° C. for 1 hour. TheProtein G Sepharose was washed six times with the lysis buffer (1 mL),and then resuspended in 1×SDS sample buffer (Bio-Rad Laboratories,Inc.). After the suspension was treated at 100° C. for 5 minutes, thesolution was collected as a sample for SDS-PAGE (polyacrylamide gelelectrophoresis).

2-2) SDS-PAGE and Western Blotting

The SDS-PAGE sample prepared in 2-1) was stacked with SuperSep Ace 5-20%(Wako Pure Chemical Industries, Ltd.) at 50 mV for 30 minutes, and thensubjected to electrophoresis at 200 mV for 1 hour, and blotted from thegel onto a membrane at 12 mV for 47 minutes. The membrane was washedwith PBS-T (PBS (−)-0.02% Tween 20), and then blocked for 1 hour. Themembrane was washed three times with PBS-T for 5 minutes, and thenreacted with a Streptavidin-horseradish peroxidase conjugate (GEHealthcare; 2000-fold diluted with PBS-T in use) for 1 hour. Themembrane was washed twice with PBS-T for 5 minutes, and a targeted bandwas then detected by using an enhanced chemiluminescence (ECL) method. Aband indicating a molecular weight of 18 kDa was detected for any of thecase with the 218B1 antibody and the case with the 218C7 antibody,regardless of the presence or absence of DTT added. 2-3) Massspectrometry of immunoprecipitated product of cell protein with 218B1antibody and 218C7 antibody

2×10⁷ NTERA-2 cells were collected and washed twice with PBS. The cellswere collected by using a cell scraper, and centrifuged at 1500 rpm for5 minutes. After the supernatant was removed, the cells were resuspendedin 2 mL of the lysis buffer, and treated at 4° C. for 30 minutes. Theresultant was centrifuged (13000 rpm, 20 minutes, 4° C.) to prepare acell lysate. Protein G Sepharose/lysis buffer (50% slurry; 180 μL) wasadded to the cell lysate, and the resultant was rotated at 4° C. for 30minutes and then centrifuged at 4° C. for 1 hour, and the supernatantwas collected. The 218B1 antibody (about 9 μg) was added to thesupernatant, and the resultant was rotated at 4° C. for 30 minutes, towhich Protein G Sepharose/lysis buffer (50% slurry; 180 μL) was thenadded, and the resultant was rotated at 4° C. for 1 hour. The Protein GSepharose was washed six times with the lysis buffer (1 mL), and thenresuspended in 1×SDS sample buffer. After the suspension was treated at100° C. for 5 minutes, the solution was collected as a sample forSDS-PAGE. SDS-PAGE was carried out in the same manner as in 2-2), andthe electrophoresis gel was stained with CBB. The part corresponding to18 kDa was cut out of the electrophoresis gel, and subjected to massspectrometry. The mass spectrometry found that the gel piece containedclaudin-6.2-4) FACS Analysis

Since the antigen for the 218B1 antibody and 218C7 antibody wasestimated to be claudin-6 from the mass spectrometry, forced expressionanalysis by cDNA transfection was carried out. FACS analysis resultsshowed that the 218B1 antibody and 218C7 antibody exhibited strongpositive reaction for human claudin-6-expressing CHO-K1 cells,demonstrating that the antigen for the 218B1 antibody and 218C7 antibodyis claudin-6.

Example 132: Purification of Antibody from Hybridoma Culture Supernatant

The mouse anti-CLDN6 antibody B1-producing hybridoma (218B1) and mouseanti-CLDN6 antibody C7-producing hybridoma (218C7) produced in Example131 were cultured in Hybridoma-SFM (Thermo Fisher Scientific) containing10% Fetal Bovine Serum, Ultra-Low IgG (Thermo Fisher Scientific). Theculture supernatant was collected by centrifugation, and filteredthrough a filter of 0.45 μm (produced by Corning Incorporated). Theantibody was purified from the culture supernatant through rProtein Aaffinity chromatography (at 4 to 6° C.) in one step. The step of bufferdisplacement after rProtein A affinity chromatography was carried out at4 to 6° C. First, the culture supernatant was applied to a column packedwith MabSelectSuRe (produced by GE Healthcare Bioscience) equilibratedwith PBS. After the culture solution completely entered the column, thecolumn was washed with PBS in an amount twice or more the column volume.Subsequently, elution was carried out with a 2 M solution of argininehydrochloride (pH 4.0), and a fraction containing the antibody wascollected. The fraction was subjected to liquid displacement to PBS (−)by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).Finally, the fraction was concentrated with a Centrifugal UF FilterDevice VIVASPIN20 (molecular weight cutoff: UF10K, Sartorius AG, at 4°C.) to adjust the IgG concentration to 1 mg/mL or more. The fraction wasfiltered through a Minisart-Plus filter (Sartorius AG), and theresultant was used as a purified sample.

Example 133: In Vitro Evaluation of Mouse Anti-CLDN6 Antibodies B1 andC7

133-1: Evaluation of Binding Ability of Mouse Anti-CLDN6 Antibodies byFlow Cytometry

Binding activity of the mouse anti-CLDN6 antibodies produced in Example132 to human CLDN6 and its family molecules, CLDN3, CLDN4, and CLDN9,was evaluated by using a flow cytometry method. Human CLDN3/pCMV6-Entry,human CLDN4/pCMV6-Entry, human CLDN6/pCMV-Entry, humanCLDN9/pCMV6-Entry, or pCMV6-Entry purchased from OriGene Technologies,Inc. was transiently transferred into 293T cells (Thermo FisherScientific, HCL4517) by using Lipofectamine 2000 (Thermo FisherScientific), and the cells were cultured under conditions of 37° C. and5% CO₂ overnight, and then a cell suspension was prepared. Thetransfected 293T cell suspension was centrifuged to remove thesupernatant, and a mouse anti-CLDN6 antibody (clone number: B1 or C7) ora mouse IgG1 control antibody (R&D Systems, Inc.) was then added andsuspended to a final concentration of 30 μg/mL, 10 μg/mL, 3.3 μg/mL, or1.1 μg/mL, and the resultant was left to stand at 4° C. for 1 hour. Thecells were washed twice with Dulbecco's phosphate buffered saline(Sigma-Aldrich Co. LLC) containing 5% fetal bovine serum (Hyclone)(hereinafter, referred to as 5% FBS-containing PBS), andFLUORESCEIN-CONJUGATED GOAT IGG FRACTION TO MOUSE IGG (WHOLE MOLECULE)(MP Biomedicals, Inc.) 500-fold diluted with 5% FBS-containing PBS wasthen added thereto, and the cells were suspended and left to stand at 4°C. for 1 hour. After washing twice with 5% FBS-containing PBS, detectionwas carried out by using a flow cytometer (FC500; Beckman Coulter,Inc.). Data analysis was carried out by using FlowJo (Tree Star, Inc.).To confirm each transfection, the cells were permeabilized with 0.25%Tween 20-containing PBS, and then a mouse anti-FLAG antibody(Sigma-Aldrich Co. LLC) was used. FIG. 40 shows the results. In eachgraph in FIG. 40 , the ordinate represents FITC fluorescence intensityindicating the amount of the binding antibody and the abscissarepresents antibody concentrations. The mouse anti-CLDN6 antibodiesproduced bound to human CLDN6 and human CLDN9 to a similar degree, anddid not bind to human CLDN3 or human CLDN4. The mouse control IgG1 didnot bind to any of the cells.

133-2: Internalization Activity of Antibodies

Internalization activity of the mouse anti-CLDN6 antibodies B1 and C7was evaluated by using the anti-mouse IgG reagent, to which a toxin thatinhibits protein synthesis (saporin) had been conjugated, Mab-ZAP(Advanced Targeting Systems). In this evaluation, Mab-ZAP isincorporated into cells in a manner depending on the internalizationactivity of a mouse anti-CLDN6 antibody, and saporin, which inhibitsprotein synthesis, is released in the cells to suppress cell growth.

JEG-3 (ATCC HTB-36), a human choriocarcinoma cell line of humanCLDN6-positive cells, NIH:OVCAR-3 (ATCC HTB-161), a human ovarian cancercell line of human CLDN6-positive cells, or BxPC-3 (ATCC CRL-1687), ahuman pancreatic cancer cell line of human CLDN6-negative cells, wasseeded in a 96-well cell culture microplate at 2×10³ cells/well, andcultured under conditions of 37° C. and 5% CO₂ overnight. On the nextday, a mixed solution obtained by mixing each mouse anti-CLDN6 antibodyor mouse IgG1 antibody (R&D Systems, Inc.) to a final concentration of 1nM, with Mab-ZAP (final concentration: 0.5 nM) or AffiniPure GoatAnti-Mouse IgG, Fcγ Fragment Specific (Jackson ImmunoResearchLaboratories Inc.) (final concentration: 0.5 nM), without conjugatedtoxin, was added, and the cells were cultured under conditions of 37° C.and 5% CO₂ for 5 days. The number of surviving cells was determinedthrough quantification of ATP activity by using CellTiter-GloLuminescent Cell Viability Assay (Promega Corporation). The cellgrowth-suppressing effect by addition of each anti-CLDN6 antibody wasdetermined as a relative survival rate to the value for the well withoutthe mixed solution as 100%. FIG. 41 shows the results. The mouseanti-CLDN6 antibodies (B1, C7) were found to have cellgrowth-suppressing effect on the human CLDN6-positive cell lines JEG-3and NIH:OVCAR-3. On the other hand, they were found to have no cellgrowth-suppressing effect on the human CLDN6-negative cell line BxPC-3.The mouse IgG1 antibody was found to have no cell growth-suppressingeffect on any of the cell lines. These results suggest that theanti-CLDN6 antibodies (B1, C7) produced have internalization activityand are each suitable as an antibody for antibody-drug conjugates.

Example 134: Nucleotide Sequencing of cDNA Encoding Variable Region ofEach of Mouse Anti-CLDN6 Antibodies B1 and C7

134-1: Nucleotide Sequencing of cDNA Encoding Variable Region of B1Antibody

134-1-1: Preparation of Total RNA of B1 Antibody-Producing Hybridoma

To amplify cDNA encoding the variable region of the B1 antibody, totalRNA was prepared from the B1 antibody-producing hybridoma by usingTRIzol Reagent (Ambion).

134-1-2: Amplification and Sequencing of cDNA Encoding Light ChainVariable Region of B1 Antibody Through 5′-RACE PCR

Amplification of cDNA encoding the light chain variable region wascarried out by using about 1 μg of the total RNA prepared in Example134-1-1 and a SMARTer RACE 5′/3′ Kit (Clontech). As a primer to amplifycDNA encoding the variable region of the light chain gene of the B1antibody through PCR, UPM (Universal Primer A Mix: attached to theSMARTer RACE 5/3′ Kit) and a primer designed on the basis of thesequence of a known mouse light chain constant region were used.

The cDNA encoding the variable region of the light chain amplifiedthrough 5′-RACE PCR was cloned into a plasmid, and subsequently sequenceanalysis was carried out for the nucleotide sequence of the cDNAencoding the variable region of the light chain.

The determined nucleotide sequence of the cDNA encoding the variableregion of the light chain of the B1 antibody is represented by SEQ IDNO: 18, and the corresponding amino acid sequence is represented by SEQID NO: 19.

134-1-3: Amplification and Sequencing of cDNA Encoding Heavy ChainVariable Region of B1 Antibody Through 5′-RACE PCR

Amplification of cDNA encoding the heavy chain variable region wascarried out by using about 1 μg of the total RNA prepared in Example134-1-1 and a SMARTer RACE 5′/3′ Kit (Clontech). As a primer to amplifycDNA encoding the variable region of the heavy chain gene of the LB1antibody through PCR, UPM (Universal Primer A Mix: attached to theSMARTer RACE 5′/3′ Kit) and a primer designed on the basis of thesequence of a known mouse heavy chain constant region were used.

The cDNA encoding the variable region of the heavy chain amplifiedthrough 5′-RACE PCR was cloned into a plasmid, and subsequently sequenceanalysis was carried out for the nucleotide sequence of the cDNAencoding the variable region of the heavy chain.

The determined nucleotide sequence of the cDNA encoding the variableregion of the heavy chain of the B1 antibody is represented by SEQ IDNO: 20, and the corresponding amino acid sequence is represented by SEQID NO: 21.

134-2: Nucleotide Sequencing of cDNA Encoding Variable Region of C7Antibody

Nucleotide sequencing was carried out in the same manner in Example134-1. The determined nucleotide sequence of the cDNA encoding thevariable region of the light chain of the C7 antibody is represented bySEQ ID NO: 22, and the corresponding amino acid sequence is representedby SEQ ID NO: 23. The nucleotide sequence of the cDNA encoding thevariable region of the heavy chain of the C7 antibody is represented bySEQ ID NO: 24, and the corresponding amino acid sequence is representedby SEQ ID NO: 25.

Example 135: Production of Chimeric Anti-CLDN6 Antibody chB1

135-1: Construction of Expression Vector for Chimeric Anti-CLDN6Antibody chB1

135-1-1: Construction of Expression Vector pCMA-LK for Chimeric andHumanized Light Chains

About 5.4 kb of a fragment obtained by digesting the plasmidpcDNA3.3-TOPO/LacZ (Invitrogen) with the restriction enzymes XbaI andPmeI was linked to a DNA fragment including a DNA sequence encoding thehuman light chain signal sequence and human κ chain constant region, asrepresented by SEQ ID NO: 26, by using an In-Fusion HD PCR Cloning Kit(Clontech) to prepare pcDNA3.3/LK. A neomycin expression unit wasremoved from the pcDNA3.3/LK to construct pCMA-LK.

135-1-2: Construction of Expression Vector pCMA-G1LALA for Chimeric andHumanized IgG1LALA-Type Heavy Chains

A DNA fragment obtained by digesting the pCMA-LK with XbaI and PmeI toremove the light chain signal sequence and human K chain constant regionwas linked to a DNA fragment including a DNA sequence encoding the humanheavy chain signal sequence and human IgG1LALA constant region, asrepresented by SEQ ID NO: 27, by using an In-Fusion HD PCR Cloning Kit(Clontech) to construct pCMA-G1LALA.

135-1-3: Construction of Chimeric chB1 Heavy Chain Expression Vector

The DNA fragment consisting of nucleotide residues 36 to 440 of thenucleotide sequence for the chB1 heavy chain, as represented by SEQ IDNO: 33, was synthesized (GeneArt). The pCMA-G1LALA was cleaved with therestriction enzyme BlpI, and the synthesized DNA fragment was insertedinto the cleaved portion by using an In-Fusion HD PCR Cloning Kit(Clontech) to construct a chB1 heavy chain expression vector. The aminoacid sequence of the chB1 heavy chain is represented by SEQ ID NO: 32.

135-1-4: Construction of Chimeric chB1 Light Chain Expression Vector

A DNA fragment including a DNA sequence encoding the chB1 light chain,as represented by SEQ ID NO: 29, was synthesized (GeneArt). By using anIn-Fusion HD PCR Cloning Kit (Clontech), the synthesized DNA fragmentwas linked to a DNA fragment obtained by digesting the pCMA-LK with XbaIand PmeI for removal of the light chain signal sequence and human κchain constant region to construct a chB1 light chain expression vector.The amino acid sequence of the chB1 light chain is represented by SEQ IDNO: 28.

135-2: Production and Purification of Chimeric Anti-CLDN6 Antibody chB1

135-2-1: Production of Chimeric Antibody chB1

FreeStyle 293F cells (Invitrogen) were passaged and cultured inaccordance with the instruction manual. Into a 3 L Fernbach ErlenmeyerFlask (Corning Incorporated), 1.2×10⁹ FreeStyle 293F cells (Invitrogen)in the logarithmic growth phase were seeded, and diluted withFreeStyle293 expression medium (Invitrogen) to adjust to 2.0×10⁶cells/mL. To 40 mL of Opti-Pro SFM medium (Invitrogen), 0.24 mg of theheavy chain expression vector, 0.36 mg of the light chain expressionvector, and 1.8 mg of polyethyleneimine (Polyscience, Inc., #24765) wereadded and gently stirred, and further left to stand for 5 minutes, andthen added to the FreeStyle 293F cells. After shaking culture at 90 rpmin an incubator at 37° C. and 8% CO₂ for 4 hours, 600 mL of EX-CELL VPROmedium (SAFC Biosciences, Inc.), 18 mL of GlutaMAX I (Gibco), and 30 mLof Yeastolate Ultrafiltrate (Gibco) were added, and the resultant wassubjected to shaking culture at 90 rpm in an incubator at 37° C. and 8%CO₂ for 7 days, and the resulting culture supernatant was filteredthrough a Disposable Capsule Filter (ADVANTEC, #CCS-045-E1H). Thechimeric anti-CLDN6 antibody obtained was designated as “chB1”.

135-2-2: Purification of Chimeric Antibody chB1

The antibody was purified from the culture supernatant obtained inExample 135-2-1 through rProtein A affinity chromatography in one step.The culture supernatant was applied to a column packed withMabSelectSuRe (produced by GE Healthcare Bioscience) equilibrated withPBS, and the column was then washed with PBS in an amount twice or morethe column volume. Subsequently, elution was carried out with a 2 Msolution of arginine hydrochloride (pH 4.0), and a fraction containingthe antibody was collected. The antibody was subjected to bufferdisplacement to PBS (−) by dialysis (Thermo Scientific, Slide-A-LyzerDialysis Cassette). The fraction was concentrated with a Centrifugal UFFilter Device VIVASPIN20 (molecular weight cutoff: UF10K, Sartorius AG)to adjust the IgG concentration to 1 mg/mL or more. Finally, thefraction was filtered through a Minisart-Plus filter (Sartorius AG), andthe resultant was used as a purified sample.

Example 136: Production of Humanized Anti-CLDN6 Antibody

136-1: Design of Humanized Form of Anti-CLDN6 Antibody

136-1-1: Molecular Modeling of Variable Region of Chimeric Antibody chB1

A method known as homology modeling (Methods in Enzymology, 203, 121-153(1991)) was used for molecular modeling of the variable region of chB1.Molecular modeling was carried out by using the commercially availableprotein conformational analysis program BioLuminate (Schrodinger, Inc.)with a structure (PDB ID: 1X1W), as a template, registered in ProteinData Bank (Nuc. Acid Res. 35, D301-D303 (2007)) with high sequenceidentity to the variable regions of the heavy chain and light chain ofchB1.

136-1-2: Design of Humanized Amino Acid Sequence

chB1 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989)). The consensus sequence of human gamma chainsubgroup 1 and that of human kappa chain subgroup 1 specified in Kabatet al. (Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service National Institutes of Health, Bethesda, Md. (1991)) hadhigh identity to the framework regions of the chB1, and hence wererespectively selected as acceptors for the heavy chain and the lightchain. Donor residues to be transferred on the acceptors were selectedthrough analysis of the three-dimensional model, for example, withreference to criteria provided by Queen et al. (Proc. Natl. Acad. Sci.USA 86, 10029-10033 (1989)). Because the CDRL3 was rich in hydrophobicamino acids, a humanized light chain with mutation in the CDRL3 wasadditionally designed.

136-2: Humanization of chB1 Heavy Chain

The three heavy chains designed were designated as hH1, hH2, and hH3.The heavy chain full-length amino acid sequence of hH1 is represented bySEQ ID NO: 52 (FIG. 34 ). The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 52 is represented by SEQ ID NO: 53 (FIG. 34). The heavy chain full-length amino acid sequence of hH2 is representedby SEQ ID NO: 56 (FIG. 36 ). The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 56 is represented by SEQ ID NO: 57 (FIG. 36). The heavy chain full-length amino acid sequence of hH3 is representedby SEQ ID NO: 60 (FIG. 38 ). The nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 60 is represented by SEQ ID NO: 61 (FIG. 38).

FIG. 45 shows comparison among the amino acid sequences of chB1_H, whichis the heavy chain of the chimeric human anti-CLDN6 antibody chB1demonstrated in Example 135, and the humanized antibody heavy chainshH1, hH2, and hH3. Each “-” in the sequences of hH1, hH2 and hH3 denotesan amino acid residue identical to that of chB1_H at the position, andeach position with a letter symbol of an amino acid residue indicatesthat the amino acid residue is a substituted amino acid residue.

136-3: Humanization of chB1 Light Chain

The four light chains designed were designated as hL1, hL2, hL3, andhL4. The light chain full-length amino acid sequence of hL1 isrepresented by SEQ ID NO: 36 (FIG. 26 ). The nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 36 is represented by SEQID NO: 37 (FIG. 26 ). The light chain full-length amino acid sequence ofhL2 is represented by SEQ ID NO: 40 (FIG. 28 ). The nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 40 is represented by SEQID NO: 41 (FIG. 28 ). The light chain full-length amino acid sequence ofhL3 is represented by SEQ ID NO: 44 (FIG. 30 ). The nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 44 is represented by SEQID NO: 45 (FIG. 30 ). The light chain full-length amino acid sequence ofhL4 is represented by SEQ ID NO: 48 (FIG. 32 ). The nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 48 is represented by SEQID NO: 49 (FIG. 32 ).

FIG. 46 shows comparison among the amino acid sequences of chB1_L, whichis the light chain of the chimeric human anti-CLDN6 antibody chB1demonstrated in Example 135, and the humanized antibody light chainshL1, hL2, hL3, and hL4. Each “-” in the sequences of hL1, hL2, hL3, andhL4 denotes an amino acid residue identical to that of chB1_L at theposition, and each position with a letter symbol of an amino acidresidue indicates that the amino acid residue is a substituted aminoacid residue.

136-4: Design of Humanized Antibody with Combination of Heavy Chain andLight Chain

An antibody consisting of hH1 and hL1 is referred to as “H1L1 antibody”or “H1L1”. An antibody consisting of hH2 and hL2 is referred to as “H2L2antibody” or “H2L2”. An antibody consisting of hH1 and hL3 is referredto as “H1L3 antibody” or “H1L3”. An antibody consisting of hH2 and hL4is referred to as “H2L4 antibody” or “H2L4”. An antibody consisting ofhH3 and hL3 is referred to as “H3L3 antibody” or “H3L3”.

136-5: Production of Humanized Anti-CLDN6 Antibody

136-5-1: Construction of Humanized Heavy Chain Expression Vector

136-5-1-1: Construction of hH1 Expression Vector

The DNA fragment consisting of nucleotide residues 36 to 440 of thenucleotide sequence of SEQ ID NO: 53 for hH1 was synthesized (GeneArt).An hH1 expression vector was constructed in the same manner as inExample 135-1-3.

136-5-1-2: Construction of hH2 Expression Vector

The DNA fragment consisting of nucleotide residues 36 to 440 of thenucleotide sequence of SEQ ID NO: 57 for hH2 was synthesized (GeneArt).An hH2 expression vector was constructed in the same manner as inExample 135-1-3.

136-5-1-3: Construction of hH3 Expression Vector

The DNA fragment consisting of nucleotide residues 36 to 440 of thenucleotide sequence of SEQ ID NO: 61 for hH2 was synthesized (GeneArt).An hH3 expression vector was constructed in the same manner as inExample 135-1-3.

136-5-2: Construction of Humanized Light Chain Expression Vector

136-5-2-1: Construction of hL1 Expression Vector The DNA fragmentconsisting of nucleotide residues 37 to 402 of the nucleotide

sequence of SEQ ID NO: 37 for hL1 was synthesized (GeneArt). The pCMA-LKwas cleaved with the restriction enzyme BsiWI, and the synthesized DNAfragment was inserted into the cleaved portion by using an In-Fusion HDPCR Cloning Kit (Clontech) to construct an hL1 expression vector.

136-5-2-2: Construction of hL2 Expression Vector

The DNA fragment consisting of nucleotide residues 37 to 402 of thenucleotide sequence of SEQ ID NO: 41 for hL2 was synthesized (GeneArt).An hL2 expression vector was constructed in the same manner as inExample 136-5-2-1.

136-5-2-3: Construction of hL3 Expression Vector

The DNA fragment consisting of nucleotide residues 37 to 402 of thenucleotide sequence of SEQ ID NO: 45 for hL3 was synthesized (GeneArt).An hL3 expression vector was constructed in the same manner as inExample 136-5-2-1.

136-5-2-4: Construction of hL4 Expression Vector

The DNA fragment consisting of nucleotide residues 37 to 402 of thenucleotide sequence of SEQ ID NO: 49 for hL4 was synthesized (GeneArt).An hL4 expression vector was constructed in the same manner as inExample 136-5-2-1.

136-5-3: Preparation of Humanized Antibodies

136-5-3-1: Production of Humanized Antibodies H1L1, H2L2, H1L3, H2L4,and H3L3

They were produced in the same manner as in Example 135-2-1. H1L1, H2L2,H1L3, H2L4, and H3L3 were produced by using the combinations of a heavychain expression vector and a light chain expression vectorcorresponding to the combinations of a heavy chain and a light chainshown in Example 136-4.

136-5-3-2: Two-Step Purification of Humanized Antibodies H1L1, H2L2,H1L3, H2L4, and H3L3

The culture supernatant obtained in Example 136-5-3-1 was purified intwo steps through rProtein A affinity chromatography and ceramichydroxyapatite. The culture supernatant was applied to a column packedwith MabSelectSuRe (produced by GE Healthcare Bioscience) equilibratedwith PBS, and the column was then washed with PBS in an amount twice ormore the column volume. Subsequently, the antibody was eluted with a 2 Msolution of arginine hydrochloride (pH 4.0). A fraction containing theantibody was subjected to buffer displacement to PBS by dialysis (ThermoScientific, Slide-A-Lyzer Dialysis Cassette), 5-fold diluted with abuffer of 5 mM sodium phosphate/50 mM MES/pH 7.0, and then applied to aceramic hydroxyapatite column (Bio-Rad Laboratories Japan, Inc.,Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with a bufferof 5 mM NaPi/50 mM MES/30 mM NaCl/pH 7.0. Linear concentration gradientelution was carried out with sodium chloride, and a fraction containingthe antibody was collected. The fraction was subjected to bufferdisplacement to HBSor (25 mM histidine/5% sorbitol, pH 6.0) by dialysis(Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody wasconcentrated with a Centrifugal UF Filter Device VIVASPIN20 (molecularweight cutoff: UF10K, Sartorius AG) to adjust the IgG concentration to50 mg/mL. Finally, the fraction was filtered through a Minisart-Plusfilter (Sartorius AG), and the resultant was used as a purified sample.

Example 137: Evaluation of Binding Ability of Humanized Anti-CLDN6Antibody by Flow Cytometry

The binding activity of the humanized anti-CLDN6 antibody produced inExample 136 to human CLDN6 and its family molecules, CLDN3, CLDN4, andCLDN9, was evaluated by using a flow cytometry method. Used were 293Tcells transiently transfected in the same manner as in Example 133-1. Tocells into which a human CLDN6 or human CLDN9 gene had been transferred,the humanized anti-CLDN6 antibody H L1, H2L2, H1L3, H2L4, or H3L3, or ahuman IgG1 control antibody (Calbiochem) was added and suspended to afinal concentration of 100 nM, 20 nM, 4 nM, or 0.8 nM, and the resultantwas left to stand at 4° C. for 30 minutes. To cells into which a humanCLDN3 or human CLDN4 gene, or an empty vector had been transferred, thehumanized anti-CLDN6 antibody H1L1, H2L2, H1L3, H2L4, or H3L3 was addedand suspended to a final concentration of 100 nM, and the resultant wasleft to stand at 4° C. for 30 minutes. The cells were washed withDulbecco's phosphate buffered saline (Sigma-Aldrich Co. LLC) containing5% fetal bovine serum (Hyclone) (hereinafter, referred to as 5%FBS-containing PBS), and FITC AffiniPureF (ab′)2 Fragment GoatAnti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories Inc.) 150-folddiluted with 5% FBS-containing PBS was then added thereto, and the cellswere suspended and left to stand at 4° C. for 30 minutes. After washingwith 5% FBS-containing PBS, detection was carried out by using a flowcytometer (FC500; Beckman Coulter, Inc.). Data analysis was carried outby using FlowJo (Tree Star, Inc.), and mean fluorescence intensity (MFI)of FITC, which indicates the amount of the binding antibody, wascalculated. FIG. 42 shows the results. In each graph in FIG. 42 , theabscissa represents antibody concentrations and the ordinate representsMFI. The humanized anti-CLDN6 antibody produced bound to human CLDN6 andhuman CLDN9 to a similar degree, and did not bind to human CLDN3 orhuman CLDN4. The human control IgG1 did not bind to any of the cells.

Example 138: Production of Trastuzumab Variant

138-1: Construction of Heavy Chain Expression Vector forTrastuzumab-LALA

The DNA fragment consisting of nucleotide residues 36 to 434 of thenucleotide sequence of SEQ ID NO: 74 for the heavy chain oftrastuzumab-LALA was synthesized (GeneArt). An expression vector wasconstructed in the same manner as in Example

135-1-3. The Amino Acid Sequence of the Heavy Chain of Trastuzumab-LALAis Represented by SEQ ID NO: 75.

138-2: Construction of Light Chain Expression Vector forTrastuzumab-LALA

The DNA fragment consisting of nucleotide residues 37 to 402 of thenucleotide sequence of SEQ ID NO: 72 for the light chain oftrastuzumab-LALA was synthesized (GeneArt). An expression vector wasconstructed in the same manner as in Example

136-5-2-1. The Amino Acid Sequence of the Light Chain ofTrastuzumab-LALA is Represented by SEQ ID NO: 73.

138-3: Production of Trastuzumab Variant

A trastuzumab variant was produced in the same manner as in Example135-2-1.

138-4: Purification of Trastuzumab Variant

Trastuzumab-LALA was purified from the culture supernatant obtained inExample 138-3 in the same manner as in Example 135-2-2, except thatbuffer displacement was carried out not to PBS (−) but to 50 mMphosphate buffer solution (pH 6.0).

Example 139: Sugar Chain Remodeling (Trastuzumab Variant-MSG1) Step 1:(Fucα1,6)GlcNAc-trastuzumab variant

The operation in step 1 of Example 58 was carried out with a ca. 22.3mg/mL trastuzumab variant solution (50 mM phosphate buffer (pH 6.0))(2.7 mL) prepared in Example 138 to afford a 6.1 mg/mL(Fucα1,6)GlcNAc-trastuzumab variant solution (50 mM phosphate buffer (pH6.0)) (6.1 mL).

Step 2: Trastuzumab Variant-[MSG1-N₃]₂

The operation in step 1 in Example 60 was carried out with the 6.1 mg/mL(Fucα1,6)GlcNAc-trastuzumab variant solution (50 mM phosphate buffer (pH6.0)) (6.1 mL) obtained in step 1 to afford a 10.2 mg/mL trastuzumabvariant-[MSG1-N3]₂ solution (phosphate buffered saline (pH 6.0)) (3.7mL).

Example 140: ADC55

(The compound to be obtained in step 1 has geometric isomers of thetriazole ring as illustrated in this formula, and the compound obtainedin step 1 of Example 140 retains a linker as a mixture of the twostructures shown above as R.)

Step 1: Conjugation of Antibody and Drug-Linker

To a solution of the antibody obtained in step 2 of Example 139 inphosphate buffered saline (pH 6.0) (10.2 mg/mL, 0.40 mL), a solution ofphosphate buffered saline (pH 6.0) (0.40 mL), 1,2-propanediol (0.767mL), dimethylformamide (0.20 mL), and a 10 mM dimethylformamide solutionof the compound obtained in step 13 of Example 3 (0.033 mL; 12equivalents per antibody molecule) were added at room temperature, andthe resultant was reacted at room temperature with a tube rotator(MTR-103, AS ONE Corporation) for 48 hours.

Purification operation: The solution was purified by using commonoperation D to afford 7.00 mL of a solution containing the targetedcompound.

Characterization: The following characteristic values were obtained byusing common operations E and F.

Antibody concentration: 0.39 mg/mL, antibody yield: 1.38 mg (35%),average number of conjugated drug molecules per antibody molecule (n):1.8

Example 141: Anticellular Effect of Anti-HER2 Antibody-Drug Conjugate

KPL-4 cells (Dr. Junichi Kurebayashi, Kawasaki Medical School), a humanbreast cancer cell line of HER2 antigen-positive cells, were preparedwith RPMI1640 Medium (Thermo Fisher Scientific; hereinafter, referred toas RPMI medium) containing 10% fetal bovine serum (Hyclone) to reach6.25×10³ cells/mL, and 80 μL portions of them were added to a 96-wellcell culture microplate. After addition of the cells, the cells werecultured at 37° C. and 5% CO2 overnight.

On the next day, 20 μL portions of an anti-HER2 antibody-drug conjugatediluted with RPMI medium to 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032nM, 6.4 pM, 1.3 pM, and 0.26 pM were added to the microplate. To eachwell without any antibody-drug conjugate, 20 μL of RPMI medium wasadded. KPL-4 was cultured at 37° C. and 5% CO₂ for 6 days. Afterculturing, the microplate was taken out of the incubator, and left tostand at room temperature for 30 minutes. CellTiter-Glo Luminescent CellViability Assay (Promega Corporation) in an amount equivalent to that ofthe culture solution was added, and stirred using a plate mixer. Themicroplate was left to stand at room temperature for 10 minutes, andthereafter the amount of emission was measured by using a plate reader(PerkinElmer). Cell survival rates were calculated in the same manner asin Example 123.

The anti-HER2 antibody-drug conjugates ADC49 and ADC55 each exhibited ananticellular effect of 0.001<IC₅₀<0.01 (nM) on the KPL-4 cells.

Example 142: Antitumor Test for Anti-HER2 Antibody-Drug Conjugate (1)

Antitumor effect of the anti-HER2 antibody-drug conjugates was measuredby using the same experiment animals and method as in Example 124.

KPL-4 cells (Dr. Junichi Kurebayashi, Kawasaki Medical School) weresuspended in Dulbecco's phosphate buffered saline (Sigma-Aldrich Co.LLC), and 1.5×10⁷ cells were subcutaneously transplanted to the rightflank of each female nude mouse (Day 0), and the mice were randomlygrouped on Day 14. The anti-HER2 antibody-drug conjugate ADC49 or theanti-HER2 antibody-drug conjugate ADC55 was administered into the tailvein on Day 14 at a dose of 0.33 mg/kg. As a control group (Vehiclegroup), ABS buffer was administered in the same manner.

FIG. 47 shows the results. ADC49 and ADC55 exhibited comparableantitumor activity with administration of 0.33 mg/kg. No weight losscaused by administration of ADC49 or ADC55 was found for the mice.

Example 143: Antitumor Test for Anti-HER2 Antibody-Drug Conjugate (2)

JIMT-1 cells (DSMZ ACC 589) were suspended in physiological saline(Otsuka Pharmaceutical Factory, Inc.), and 5×10⁶ cells weresubcutaneously transplanted to the right flank of each female nude mouse(Day 0), and the mice were randomly grouped on Day 11. ADC55 wasadministered into the tail vein on Day 11 at a dose of 0.4 mg/kg or 0.2mg/kg. As a control group (Vehicle group), ABS buffer was administeredin the same manner.

FIG. 48 shows the results. ADC55 was found to have strong antitumoreffect causing regression of tumor in administration of 0.4 mg/kg. Noweight loss caused by administration of ADC55 was found for the micewith any dose of administration.

Example 144: Antitumor Test for Anti-HER2 Antibody-Drug Conjugate (3)

CFPAC-1 cells (ATCC CRL-1918) were suspended in physiological saline(Otsuka Pharmaceutical Factory, Inc.), and 5×10⁶ cells weresubcutaneously transplanted to the right flank of each female nude mouse(Day 0), and the mice were randomly grouped on Day 10. The anti-HER2antibody-drug conjugate ADC49 or ADC55, or the anti-LPS antibody-drugconjugate ADC53 was administered into the tail vein on Day 10 at a doseof 0.4 mg/kg. As a control group (Vehicle group), ABS buffer wasadministered in the same manner.

FIG. 49 shows the results. Strong antitumor effect causing regression oftumor was found for any mice to which ADC49 or ADC55 had beenadministered. No weight loss caused by administration of ADC49, ADC55,or ADC53 was found for the mice.

Example 145: Drug-Linker 43

To a solution of the compound obtained in step 12 of Example 3 (0.051 g,0.049 mmol) and commercially available maleimidocaproic acid (0.011 g,0.054 mmol) in dichloromethane (5 mL),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.010 g,0.054 mmol) was added at room temperature, and the resultant was stirredat room temperature for 2 hours. After the reaction solution was dilutedwith chloroform, the organic layer was washed with water and dried oversodium sulfate. After the organic solvent was concentrated, theresulting residue was purified by silica gel chromatography(chloroform:methanol=97.5:2.5 (v/v) to 90:10 (v/v)) to afford thedesired compound (41 mg, 69%).

¹H-NMR (DMSO-D₆) δ: 9.94 (1H, s), 8.20-8.12 (1H, m), 7.86-7.77 (1H, m),7.65-7.52 (2H, m), 7.45 (1H, s), 7.38 (2H, d, J=7.9 Hz), 7.30 (1H, s),7.24-7.16 (2H, m), 7.11-7.01 (1H, m), 7.00 (2H, s), 6.91 (2H, d, J=8.5Hz), 6.83-6.67 (1H, m), 6.63-6.45 (2H, m), 6.31 (1H, s), 5.81-5.71 (1H,m), 5.23-5.14 (1H, m), 4.86-4.74 (1H, m), 4.43-4.32 (1H, m), 4.24-4.11(2H, m), 4.03-3.88 (3H, m), 3.88-3.69 (4H, m), 3.76 (3H, s), 3.66 (3H,s), 3.60-3.49 (2H, m), 3.47-3.08 (5H, m), 2.82-2.64 (1H, m), 2.40-2.29(1H, m), 2.25-2.04 (2H, m), 2.04-1.89 (1H, m), 1.88-1.67 (4H, m),1.63-1.38 (7H, m), 1.35-1.09 (6H, m), 0.90-0.76 (6H, m), 0.76-0.50 (4H,m).

MS (APCI, ESI) m/z: 1224 (M+H)⁺

An anti-CLDN6 antibody (Example 136 (H1L1))-PBD ADC (cysteineconjugation, m¹=2) of drug-linker 43 and drug-linker 44 (Example 146)was produced by using a known method (WO 2014/057687). The ADC exhibiteda strong antitumor effect.

Example 146: Drug-Linker 44

The compound obtained in step 12 of Example 3 (0.047 g, 0.046 mmol) andcommercially availableN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycine (0.016g, 0.050 mmol) were reacted in the same manner as in Example 145 toafford the desired compound (30 mg, 50%).

¹H-NMR (DMSO-D₆) δ: 9.92 (1H, s), 8.23 (1H, d, J=6.7 Hz), 8.12-8.02 (2H,m), 7.84 (1H, d, J=9.1 Hz), 7.65-7.50 (2H, m), 7.45 (1H, s), 7.38 (2H,d, J=9.1 Hz), 7.30 (1H, s), 7.26-7.16 (2H, m), 7.10-7.02 (1H, m), 7.00(2H, s), 6.91 (2H, d, J=9.1 Hz), 6.82-6.67 (1H, m), 6.62-6.48 (2H, m),6.31 (1H, s), 5.80-5.72 (1H, m), 5.25-5.15 (1H, m), 4.85-4.77 (1H, m),4.43-4.30 (1H, m), 4.25-4.14 (2H, m), 4.06-3.87 (3H, m), 3.86-3.71 (6H,m), 3.76 (3H, s), 3.71-3.62 (2H, m), 3.66 (3H, s), 3.61-3.45 (2H, m),3.45-3.08 (1H, m), 2.81-2.64 (2H, m), 2.39-2.29 (1H, m), 2.14-2.04 (2H,m), 2.05-1.90 (1H, m), 1.90-1.68 (1H, m), 1.62-1.39 (7H, m), 1.35-1.26(6H, m), 1.26-1.13 (6H, m), 0.91-0.77 (6H, m), 0.75-0.52 (4H, m).

MS (APCI, ESI) m/z: 1338 (M+H)⁺

Example 147: Drug-Linker 45

The compound obtained in step 12 of Example 3 (0.050 g, 0.049 mmol) andcommercially available31-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azahentriacontan-1-oicacid (0.029 g, 0.049 mmol) were reacted in the same manner as in Example145 to afford the desired compound (45 mg, 57%).

MS (APCI, ESI) m/z: 1604 (M+H)⁺

Example 148: Drug-Linker 46

The compound obtained in step 9 of Example 15 (0.081 g, 0.085 mmol) andcommercially availableN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycine (0.042g, 0.13 mmol) were reacted in the same manner as in step 1 of Example 16to afford the desired compound (0.089 g, 82%).

MS (APCI, ESI) m/z: 1257 (M+H)⁺

Example 149: Drug-Linker 47

To a solution of commercially availableN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycine (0.036g, 0.11 mmol) in dichloromethane (10 mL),N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.035 g, 0.14 mmol) wasadded at room temperature, and the resultant was stirred at roomtemperature for 1 hour. To the reaction solution, the compound obtainedin step 11 of Example 4 (0.10 g, 0.10 mmol) and methanol (1 mL) wereadded, and the resultant was stirred at room temperature for 18 hours.After the reaction solution was concentrated, the resulting residue waspurified by silica gel chromatography(chloroform-chloroform:methanol=80:20 (v/v)) to afford the desiredcompound (0.078 g, 60%).

MS (APCI, ESI) m/z: 1309 (M+H)⁺

Example 150: Drug 11

Steps 1 to 4

The compound obtained in step 7 of Example 19 was reacted in the samemanner as in step 2 of Example 3, step 10 of Example 3, step 11 ofExample 3, and step 12 of Example 3 to afford drug 11.

¹H-NMR (CDCl₃) δ: 7.89-7.80 (1H, m), 7.60-7.51 (2H, m), 7.43-7.40 (1H,m), 7.34-7.30 (2H, m), 6.91-6.88 (2H, m), 6.81 (1H, d, J=3.0 Hz),4.39-4.32 (1H, m), 4.11-4.06 (4H, m), 3.95 (4H, d, J=3.0 Hz), 3.87-3.84(5H, m), 3.68-3.61 (2H, m), 3.51-3.49 (3H, m), 3.44-3.34 (2H, m), 2.53(1H, dd, J=13.0, 8.2 Hz), 2.01-1.96 (4H, m), 1.69-1.68 (2H, m),1.30-1.25 (1H, m), 0.93-0.77 (4H, m).

MS (APCI, ESI) m/z: 691 (M+H)⁺.

Example 151: Drug 12

Step 1

The compound obtained in step 3 of Example 47 (0.77 g, 2.8 mmol) wasdissolved in dichloromethane (20 mL), and pyridine (0.338 mL, 4.20 mmol)and allyl chloroformate (0.355 mL, 3.36 mmol) were added thereto at 0°C., and the resultant was stirred at room temperature for 2 hours. Afterthe reaction solution was concentrated, the resulting residue wasdissolved in methanol (20 mL), and potassium carbonate (1.93 g, 14.0mmol) was added thereto at room temperature, and the resultant wascontinually stirred for 3 hours. Water was added to the reactionsolution, and methanol was distilled off under reduced pressure, and 1 Nhydrochloric acid was added to the resulting residue, which wasextracted with chloroform. The organic layer was dried over sodiumsulfate, and filtered, and the solvent was then distilled off underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography [hexane:ethyl acetate=100:0 (v/v) to 0:100 (v/v)]to afford the desired compound (0.921 g, 92%) as a pale yellow solid.

MS (APCI, ESI) m/z: 359 (M+H)⁺.

Steps 2 to 4

The compound obtained in step 1 was reacted in the same manner as insteps 10 to 12 of Example 3 to afford drug 12.

¹HNMR (CDCl₃) δ: 7.89 (1H, d, J=4.2 Hz), 7.59 (1H, s), 7.53 (1H, s),7.40 (1H, s), 7.34 (2H, d, J=8.5 Hz), 6.91 (2H, d, J=8.5 Hz), 6.82 (1H,s), 6.05 (1H, s), 4.44-4.39 (1H, m), 4.16-4.11 (2H, m), 4.09-4.04 (2H,m), 3.99 (2H, t, J=6.7 Hz), 3.95 (3H, s), 3.84 (6H, s), 3.72 (1H, d,J=12.1 Hz), 3.59-3.49 (3H, m), 3.45-3.34 (2H, m), 2.00-1.92 (4H, m),1.79 (1H, dd, J=12.4, 7.0 Hz), 1.70-1.64 (2H, m), 1.25 (1H, t, J=7.0Hz), 0.73-0.55 (4H, m).

MS (APCI, ESI) m/z: 693 (M+H)⁺.

Example 152: Drugs 13 to 16

[Cyclobutyl derivative: drug 13] n=1, R═F

Step 1

To a solution of commercially availableN-T-BOC-4-(3,3-difluorocyclobutyl)-L-proline (OmegaChem Inc., OC-0707)(12 g, 41 mmol) in methanol (200 mL), thionyl chloride (10 mL, 138 mmol)was slowly added dropwise at −78° C. (dry ice-acetone bath). After thedropwise addition, the refrigerant bath was removed, and the reactionmixture was stirred at room temperature overnight. The reaction mixturewas concentrated under reduced pressure, and the residue was washed withdiethyl ether to afford the desired compound (10 g, quant.).

[Step 2]

To a suspension solution of the compound obtained in step 1 (10 g, 41.4mmol) and sodium carbonate (8.77 g, 82.7 mmol) in 1,4-dioxane (200 mL)and water (50 mL), benzyl chloroformate (8.82 mL, 62.0 mmol) was slowlyadded under ice-cooling. After the completion of the reaction, water wasadded thereto, and the reaction mixture was extracted with ethylacetate. The organic layer was washed with water and brine, and driedover anhydrous sodium sulfate. After filtration, the solvent wasdistilled off under reduced pressure to afford the desired compound.

Steps 3 to 15

The compound obtained in the previous step was reacted in the samemanner as in steps 1 to 5 of Example 1 and steps 1 to 8 of Example 45 toafford drug 13.

¹H NMR(CDCl₃) δ: 7.72-7.71 (1H, m), 7.53-7.49 (3H, m), 7.34-7.28 (2H,m), 6.91-6.78 (3H, m), 6.06-6.03 (1H, m), 4.35-4.28 (1H, m), 4.16-4.04(2H, m), 4.02-3.96 (1H, m), 3.94 (3H, s), 3.90-3.76 (2H, m), 3.84 (2H,s), 3.82 (3H, s), 3.59-3.31 (2H, m), 2.76-2.44 (8H, m), 1.99-1.88 (4H,m), 1.71-1.59 (4H, m).

MS (APCI, ESI) m/z: 743 (M+H)⁺.

[Cyclobutyl derivative: drug 14] n=1, R═H

Steps 2 to 15

Commercially available 4-cyclobutyl-L-proline methyl ester hydrochloride(OmegaChem Inc., OC-0728) was reacted in the same manner as in step 2,steps 1 to 5 of Example 1, and steps 1 to 8 of Example 45 to afforddrug-linker 14.

¹H NMR(CDCl₃) δ: 7.73-7.45 (3H, m), 7.34-7.14 (2H, m), 6.94-6.42 (3H,m), 6.05-5.99 (1H, m), 5.30-5.06 (2H, m), 4.49-4.28 (2H, m), 4.17-3.96(2H, m), 3.95-3.79 (9H, m), 3.77-3.30 (6H, m), 2.77-2.70 (1H, m),2.37-2.30 (1H, m), 2.19-1.85 (8H, m), 1.73-1.55 (4H, m), 1.30-1.16 (2H,m).

MS (APCI, ESI) m/z: 707 (M+H)⁺.

[Cyclopropyl derivative: drug 15] n=0, R═F, *: (S)

Commercially available N-t-BOC-4S-(2,2-difluorocyclopropyl)-L-proline(OmegaChem Inc., OC-0732) was reacted in the same manner as in steps 1to 15 to afford drug-linker 15.

¹HNMR (CDCl₃) δ: 7.76-7.75 (1H, m), 7.54-7.47 (3H, m), 7.33-7.27 (2H,m), 6.93-6.85 (2H, m), 6.83-6.79 (1H, m), 6.08-5.96 (1H, m), 4.32-4.30(1H, m), 4.16-3.96 (3H, m), 3.94 (3H, s), 3.84 (3H, s), 3.82 (3H, s),3.74-3.67 (3H, m), 3.59-3.51 (2H, m), 3.39-3.34 (1H, m), 2.76-2.69 (2H,m), 2.38-2.33 (1H, m), 1.98-1.89 (4H, m), 1.69-1.54 (4H, m), 1.30-1.22(2H, m).

MS (APCI, ESI) m/z: 729 (M+H)⁺.

[Cyclopropyl derivative: drug 16] n=0, R═F, *: (R)

Commercially available N-t-BOC-4R-(2,2-difluorocyclopropyl)-L-proline(OmegaChem Inc., OC-0722) was reacted in the same manner as in steps 1to 15 to afford drug-linker 16.

¹HNMR (CDCl₃) δ: 7.76-7.69 (1H, m), 7.55-7.44 (3H, m), 7.33-7.28 (2H,m), 6.94-6.80 (2H, m), 6.59-6.42 (1H, m), 6.08-5.97 (1H, m), 4.36-4.24(1H, m), 4.20-3.95 (3H, m), 3.95-3.76 (12H, m), 3.75-3.50 (2H, m),3.42-3.31 (1H, m), 2.79-2.52 (2H, m), 2.48-2.32 (1H, m), 2.06-1.83 (4H,m), 1.72-1.41 (4H, m), 1.28-1.15 (2H, m).

MS (APCI, ESI) m/z: 729 (M+H)⁺.

Example 153: [N₃-PEG (3)]₂-SG (10) Step 1: Fmoc-(SG-)Asn Free Form

Fmoc-(SG-)Asn (1S2S-1 INC-Asn-Fmoc, produced by GlyTech, Inc., 2 g) wasdissolved in an appropriate amount of a 0.1% aqueous solution oftrifluoroacetic acid, and subjected to separation/purification byreversed-phase HPLC in multiple separate operations. The eluent was a0.1% aqueous solution of trifluoroacetic acid and a 0.1% acetonitrilesolution of trifluoroacetic acid, the apparatus used was a Purif-Rp2(produced by Shoko Scientific Co., Ltd.), and the column used was anInertsil ODS-3 (produced by GL Sciences, Inc.). Fractions containing thedesired product UV-detected (220 nm) during the elution were collectedtogether, and freeze-dried. A colorless solid (1.8 g) was obtained.

Step 2: Synthesis of ([N₃-PEG (3)]₂-SG)-Asn-PEG (3)-N₃

To an N,N-dimethylformamide solution (10 mL) of the Fmoc-(SG-)Asn freeform prepared in step 1 (1000 mg), an N,N-dimethylformamide solution (3mL) of HATU (891 mg, 2.34 mmol) and an N,N-dimethylformamide solution (3mL) of 11-azide-3,6,9-trioxaundecane-1-amine (Tokyo Chemical IndustryCo., Ltd., 511 mg, 2.34 mmol) and diisopropylethylamine (816 μL, 4.69mmol) were added, and the resultant was stirred at 37° C. for 3 hours.Further, an N,N-dimethylformamide solution (500 μL) of HATU (148 mg,0.39 mmol) was added thereto, and the resultant was stirred at 37° C.for 1 hour. Thereafter, piperidine (386 μL, 3.91 mmol) was addedthereto, and the resultant was stirred at 37° C. for 1 hour. After thecompletion of the reaction, acetic acid (469 μL) was added thereto.

The reaction solution was halved and transferred into two jumbo conicaltubes (175 mL) to each of which diethyl ether (100 mL) had been added inadvance. The solid matter was precipitated by using a small centrifuge(Hitachi Koki Co., Ltd., CF16RX) and the supernatant was removed. Thegum-like solid matter was transferred into a centrifuge tube (50 mL),and diethyl ether (30 mL) and acetonitrile (10 mL) were added thereto,and the resultant was decanted. This operation was repeated twice. Inthe same manner, an appropriate amount of acetonitrile or an appropriateamount of diethyl ether was added and the resultant was decanted, andthen dried under reduced pressure to afford a crude product. The solidmatter obtained was dissolved in an appropriate amount of a 0.2%trifluoroacetic acid aqueous solution, and subjected toseparation/purification by reversed-phase HPLC. The eluent was a 0.1%trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acidacetonitrile solution, the apparatus used was a Purif-Rp2 (produced byShoko Scientific Co., Ltd.), and the column used was an Inertsil ODS-3(produced by GL Sciences, Inc.). Fractions containing the desiredproduct UV-detected (220 nm) during the elution were collected together,and freeze-dried to afford the titled desired compound (637 mg) as acolorless solid.

ESI-MS: Calcd for C₁₁₂H₁₉₂N₂₀O₇₀: [M+3H]³⁺ 980.6 (ave.), Found 980.4

Step 3: Synthesis of [N₃-PEG (3)]₂-SG (10)

In a 2 mL tube, ([N₃-PEG (3)]₂-SG)-Asn-PEG (3)-N₃ synthesized in step-2(78.6 mg) was dissolved in 100 mM phosphate buffer (NACALAI TESQUE,INC., 465 μL) at pH 6.0. Thereto, 1 U/mL EndoM (Tokyo Chemical IndustryCo., Ltd., 70 μL) was added, and the resultant was shaken at 28° C. for5 hours, and then left to stand at room temperature for 4 days. Afterthe completion of the reaction, an appropriate amount of a 0.2%trifluoroacetic acid aqueous solution was added thereto, and theresultant was subjected to separation/purification by reversed-phaseHPLC. The eluent was a 0.1% trifluoroacetic acid aqueous solutio and a0.1% trifluoroacetic acid acetonitrile solution, the apparatus used wasa Purif-Rp2 (produced by Shoko Scientific Co., Ltd.), and the columnused was an Inertsil ODS-3 (produced by GL Sciences, Inc.). Fractionscontaining the desired product UV-detected (220 nm) during the elutionwere collected together, and freeze-dried to afford the titled desiredcompound (40 mg) as a colorless solid.

ESI-MS: Calcd for C₉₂H₁₅₇N₁₃O₆₁: [M+2H]²⁺ 1211.7 (ave.), Found 1211.5

Example 154: [N₃-PEG (3)]-MSG2 (9), [N₃-PEG (3)]-MSG1 (9) Step 1:(MSG1-)Asn and (MSG2-)Asn

The commercially available product monosialo-Asn free(1S2G/1G2S-10NC-Asn, produced by GlyTech, Inc.) (referred to as“(MSG-)Asn”) (500 mg) was subjected to separation/purification byreversed-phase HPLC under conditions below to separate into (MSG1-)Asneluted as the 1st main peak (retention time: around 15 to 19 min) and(MSG2-)Asn eluted as the 2nd main peak (retention time: around 21 to 26min). The eluent used was a 0.1% aqueous solution of formic acid, theapparatus used was an ELS-PDA trigger preparative system (produced byJASCO Corporation), the column used was an Inertsil ODS-3 (10 um,30ϕ×250 mm, produced by GL Sciences, Inc.), and the flow rate was 30mL/min. Fractions belonging to the first peak UV-detected (210 nm)during the elution were collected together, and freeze-dried to afford(MSG1-)Asn (238 mg) as a colorless solid. Fractions belonging to thesecond peak UV-detected were collected together, and freeze-dried toafford (MSG2-)Asn (193 mg) as a colorless solid.

Step-2: Fmoc-(MSG2-)Asn Free Form

(MSG2-)Asn synthesized in step 1 (900 mg) was dissolved in anN,N-dimethylformamide solution (6 mL)/distilled water (2 mL), anddiisopropylethylamine (0.23 mL) and 9-fluorenylmethyl N-succinimidylcarbonate (223 mg) were added thereto, and the resultant was stirred atroom temperature for 30 minutes. Further, diisopropylethylamine (0.16mL), 9-fluorenylmethyl N-succinimidyl carbonate (74 mg), andN,N-dimethylformamide solution (1 mL) were added thereto, and theresultant was stirred at room temperature for 30 minutes.

The reaction solution was halved and transferred into two jumbo conicaltubes (175 mL) to each of which diethyl ether (80 mL)/acetonitrile (4mL) had been added in advance. The solid matter was precipitated byusing a small centrifuge (Hitachi Koki Co., Ltd., CF16RX) and thesupernatant was removed. The gum-like solid matter was transferred intoa centrifuge tube (50 mL), and diethyl ether (30 mL) and acetonitrile(10 mL) were added thereto, and the resultant was decanted. Thisoperation was repeated twice. In the same manner, an appropriate amountof acetonitrile or an appropriate amount of diethyl ether was added andthe resultant was decanted, and then dried under reduced pressure toafford a crude product. The solid matter obtained was dissolved in anappropriate amount of a 0.2% trifluoroacetic acid aqueous solution, andsubjected to separation/purification by reversed-phase HPLC. The eluentwas a 0.1% trifluoroacetic acid aqueous solution and a 0.1%trifluoroacetic acid acetonitrile solution, the apparatus used was aPurif-Rp2 (produced by Shoko Scientific Co., Ltd.), and the column usedwas an Inertsil ODS-3 (produced by GL Sciences, Inc.). During eluting,fractions containing the UV-detected (220 nm) desired product werecollected together, and freeze-dried to afford the titled desiredcompound (830 mg) as a colorless solid.

Step 3: ([N₃-PEG (3)]-MSG2)-Asn-PEG (3)-N₃

With use of Fmoc-(MSG2-)Asn) synthesized in step 2 (830 mg, a crudeproduct of the titled desired product (1.06 g) was obtained in the samemanner as in step 2 of Example 153. This was used for the subsequentreaction without additional purification.

Similarly, the following ([N₃-PEG (3)]-MSG1)-Asn-PEG (3)-N₃ can besynthesized from Fmoc-(MSG1-)Asn.

Step 4: [N₃-PEG (3)]-MSG2 (9)

In a 2 mL collection vial, the crude product of ([N₃-PEG(3)]-MSG2)-Asn-PEG (3)-N₃ synthesized in step 3 (150 mg) was dissolvedin 200 mM potassium phosphate buffer (750 μL) at pH 6.25 prepared with200 mM KH₂PO₄ and 200 mM KH₂PO₄. Endo-Rp (3 ug) was added thereto, andthe resultant was left to stand at 50° C. for 16 hours. After thecompletion of the reaction, a 5% aqueous solution of trifluoroaceticacid (150 μL) was added thereto, and the resultant was subjected toseparation/purification by reversed-phase HPLC. The eluent was a 0.1%aqueous solution trifluoroacetic acid aqueous solution and a 0.1%trifluoroacetic acid acetonitrile solution, the apparatus used was anELS-PDA trigger preparative system (produced by JASCO Corporation), andthe column used was an Inertsil ODS-3 (produced by GL Sciences, Inc.).Fractions containing the desired product UV-detected (210 nm) during theelution were collected together, and freeze-dried to afford the titleddesired compound (62 mg) as a colorless solid.

ESI-MS: Calcd for C₇₃H₁₂₄N₈O₅₁: [M+2H]²⁺ 965.3 (ave.), Found 965.4

Similarly, the following [N₃-PEG (3)]-MSG1 (9) can be synthesized from([N₃-PEG (3)]-MSG1)-Asn-PEG (3)-N₃.

INDUSTRIAL APPLICABILITY

Use of the antibody-drug conjugate, antibody and/or PBD derivative, andso on of the present invention enables treatment or prevention ofvarious cancers.

FREE TEXT OF SEQUENCE LISTING

SEQ ID NO: 1—Amino acid sequence of human CLDN6

SEQ ID NO: 2—Nucleotide sequence of cDNA encoding amino acid sequence ofhuman CLDN6

SEQ ID NO: 3—Amino acid sequence of human CLDN9

SEQ ID NO: 4—Nucleotide sequence of cDNA encoding amino acid sequence ofhuman CLDN9

SEQ ID NO: 5—Amino acid sequence of CDRL1 of B1 antibody light chain

SEQ ID NO: 6—Amino acid sequence of CDRL2 of B1 antibody light chain

SEQ ID NO: 7—Amino acid sequence of CDRL3 of B1 antibody light chain

SEQ ID NO: 8—Amino acid sequence of CDRL3 of humanized B1 antibody lightchain L4

SEQ ID NO: 9—Amino acid sequence of CDRH1 of B1 antibody heavy chain

SEQ ID NO: 10—Amino acid sequence of CDRH2 of B1 antibody heavy chain

SEQ ID NO: 11—Amino acid sequence of CDRH3 of B1 antibody heavy chain

SEQ ID NO: 12—Amino acid sequence of CDRL1 of C7 antibody light chain

SEQ ID NO: 13—Amino acid sequence of CDRL2 of C7 antibody light chain

SEQ ID NO: 14—Amino acid sequence of CDRL3 of C7 antibody light chain

SEQ ID NO: 15—Amino acid sequence of CDRH1 of C7 antibody heavy chain

SEQ ID NO: 16—Amino acid sequence of CDRH2 of C7 antibody heavy chain

SEQ ID NO: 17—Amino acid sequence of CDRH3 of C7 antibody heavy chain

SEQ ID NO: 18—Nucleotide sequence of cDNA encoding variable region of B1antibody light chain

SEQ ID NO: 19—Amino acid sequence of variable region of B1 antibodylight chain

SEQ ID NO: 20—Nucleotide sequence of cDNA encoding variable region of B1antibody heavy chain

SEQ ID NO: 21—Amino acid sequence of variable region of B1 antibodyheavy chain

SEQ ID NO: 22—Nucleotide sequence of cDNA encoding variable region of C7antibody light chain

SEQ ID NO: 23—Amino acid sequence of variable region of C7 antibodylight chain

SEQ ID NO: 24—Nucleotide sequence of cDNA encoding variable region of C7antibody heavy chain

SEQ ID NO: 25—Amino acid sequence of variable region of C7 antibodyheavy chain

SEQ ID NO: 26—DNA fragment including DNA sequence encoding human lightchain signal sequence and human κ chain constant region

SEQ ID NO: 27—DNA fragment including DNA sequence encoding human heavychain signal sequence and human IgG1 LALA constant region

SEQ ID NO: 28—Amino acid sequence of chB1 light chain

SEQ ID NO: 29—DNA fragment including DNA sequence encoding amino acidsequence of chB1 light chain

SEQ ID NO: 30—Amino acid sequence of variable region of chB1 light chain

SEQ ID NO: 31—Nucleotide sequence encoding chB1 light chain variableregion

SEQ ID NO: 32—Amino acid sequence of chB1 heavy chain

SEQ ID NO: 33—Nucleotide sequence encoding chB1 heavy chain

SEQ ID NO: 34—Amino acid sequence of variable region of chB1 heavy chain

SEQ ID NO: 35—Nucleotide sequence encoding variable region of chB1 heavychain

SEQ ID NO: 36—Amino acid sequence of humanized antibody light chain hL1

SEQ ID NO: 37—Nucleotide sequence encoding humanized antibody lightchain hL1

SEQ ID NO: 38—Amino acid sequence of variable region of humanizedantibody light chain hL1

SEQ ID NO: 39—Nucleotide sequence encoding variable region of humanizedantibody light chain hL1

SEQ ID NO: 40—Amino acid sequence of humanized antibody light chain hL2

SEQ ID NO: 41—Nucleotide sequence encoding humanized antibody lightchain hL2

SEQ ID NO: 42—Amino acid sequence of variable region of humanizedantibody light chain hL2

SEQ ID NO: 43—Nucleotide sequence encoding variable region of humanizedantibody light chain hL2

SEQ ID NO: 44—Amino acid sequence of humanized antibody light chain hL3

SEQ ID NO: 45—Nucleotide sequence encoding humanized antibody lightchain hL3

SEQ ID NO: 46—Amino acid sequence of variable region of humanizedantibody light chain hL3

SEQ ID NO: 47—Nucleotide sequence encoding variable region of humanizedantibody light chain hL3

SEQ ID NO: 48—Amino acid sequence of humanized antibody light chain hL4

SEQ ID NO: 49—Nucleotide sequence encoding humanized antibody lightchain hL4

SEQ ID NO: 50—Amino acid sequence of variable region of humanizedantibody light chain hL4

SEQ ID NO: 51—Nucleotide sequence encoding variable region of humanizedantibody light chain hL4

SEQ ID NO: 52—Amino acid sequence of humanized antibody heavy chain hH1

SEQ ID NO: 53—Nucleotide sequence encoding humanized antibody heavychain hH1

SEQ ID NO: 54—Amino acid sequence of variable region of humanizedantibody heavy chain hH1

SEQ ID NO: 55—Nucleotide sequence encoding variable region of humanizedantibody heavy chain hH1

SEQ ID NO: 56—Amino acid sequence of humanized antibody heavy chain hH2

SEQ ID NO: 57—Nucleotide sequence encoding humanized antibody heavychain hH2

SEQ ID NO: 58—Amino acid sequence of variable region of humanizedantibody heavy chain hH2

SEQ ID NO: 59—Nucleotide sequence encoding variable region of humanizedantibody heavy chain hH2

SEQ ID NO: 60—Amino acid sequence of humanized antibody heavy chain hH3

SEQ ID NO: 61—Nucleotide sequence encoding humanized antibody heavychain hH3

SEQ ID NO: 62—Amino acid sequence of variable region of humanizedantibody heavy chain hH3

SEQ ID NO: 63—Nucleotide sequence encoding variable region of humanizedantibody heavy chain hH3

SEQ ID NO: 64—Amino acid sequence of Trastuzumab light chain

SEQ ID NO: 65—Amino acid sequence of Trastuzumab heavy chain

SEQ ID NO: 66—Amino acid sequence of anti-LPS antibody (h #1G5-H1L1)light chain

SEQ ID NO: 67—Amino acid sequence of anti-LPS antibody (h #1G5-H1L1)heavy chain

SEQ ID NO: 68—Amino acid sequence of anti-TROP2 antibody (hRS7) lightchain

SEQ ID NO: 69—Amino acid sequence of anti-TROP2 antibody (hRS7) heavychain

SEQ ID NO: 70—Amino acid sequence of anti-CD98 antibody (hM23-H1L1)light chain

SEQ ID NO: 71—Amino acid sequence of anti-CD98 antibody (hM23-H1L1)heavy chain

SEQ ID NO: 72—Nucleotide sequence encoding Trastuzumab variant lightchain

SEQ ID NO: 73—Amino acid sequence of Trastuzumab variant light chain

SEQ ID NO: 74—Nucleotide sequence encoding Trastuzumab variant heavychain

SEQ ID NO: 75—Amino acid sequence of Trastuzumab variant heavy chain

The invention claimed is:
 1. An antibody-drug conjugate represented by[Formula 25], [Formula 26], [Formula 27], or [Formula 28], wherein:

[Formula 28] is:

wherein: m² represents 1 or 2; Ab represents an immunoglobulin G (IgG)antibody or a functional fragment of the immunoglobulin G (IgG)antibody; and the N297 glycan of Ab represents: (i) N297-(Fuc)MSG1 of[Formula 29], N297-(Fuc)MSG2 of [Formula 30], or N297-(Fuc)SG of[Formula 31]:

wherein: L(PEG) represents —NH—CH₂CH₂—(OCH₂CH₂)₃—*, wherein the —NH— atthe left end is bound via an amide bond to the carboxylic acid at the2-position of a sialic acid at the non-reducing terminal in one or bothof the 1-3 and 1-6 branched chains of the β-Man in the N297 glycan, andfurther wherein each * represents bonding to a nitrogen atom at the1-position or 3-position of the triazolyl in [Formula 25], [Formula 26],[Formula 27], or [Formula 28]; and each

represents bonding to Asn297 of the antibody; or (ii) a mixture ofN297-(Fuc)MSG1 of [Formula 29] and N297-(Fuc)MSG2 of [Formula 30] above.